Ciliary neurotrophic factor receptor antibodies

ABSTRACT

The present invention relates to the ciliary neurotropic factor (CNTF) receptor, and provides for CNTF receptor antibodies. It also relates to diagnostic techniques for identifying CNTF-related neurologic conditions.

This is a continuation of application Ser. No. 08/001,904 filed Jan. 7,1993, now abandoned, which is a continuation of application Ser. No.07/700,677 filed May 15, 1991, now abandoned; which is acontinuation-in-part of application Ser. No. 07/676,647 filed Mar. 28,1991 now U.S. Pat. No. 542,06,177; which is a continuation-in-part ofapplication Ser. No. 07/532,285 filed Jun. 1, 1990 now abandoned.

TABLE OF CONTENTS

1. Introduction

2. Background Of The Invention

2.1. Ciliary Neurotrophic Factor

2.2. Functional Properties Of Ciliary Neurotrophic Factor

2.3. Growth Factor Receptors

3. Summary Of The Invention

4. Description Of The Figures

5. Detailed Description Of The Invention

5.1. Cloning Of The Ciliary Neurotrophic Factor Receptor

5.2. Nucleic Acid Encoding Ciliary Neurotrophic Factor Receptor

5.3. Ciliary Neurotrophic Factor Receptor Peptides

5.4. Expression of Ciliary Neurotrophic Factor Receptor

5.5. Identification Of Molecules Related To The Ciliary NeurotrophicFactor Receptor

5.6. Utility Of The Invention

5.6.1. Assay Systems

5.6.2. Experimental Model Systems

5.6.2.1. Models For Increased CNTF Activity

5.6.2.2. Models For Decreased CNTF Activity

5.6.3. Diagnostic Applications

5.6.4. Therapeutic Applications

6. Example: Expression Cloning Of The Ciliary Neurotrophic FactorReceptor

6.1. Materials And Methods

6.1.1. Construction Of A CNTF-Receptor Expression Library

6.1.2. "Panning" Method

6.1.3. Identification Of Clones Containing The Ciliary NeurotrophicFactor Receptor Gene

6.1.4. Direct ¹²⁵ I-hCNTF Binding Assay

6.1.5. Fluorescence Activated Cell-Sorting Analysis

6.1.6. Iodination Of hCNTF

6.1.7. Sequencing Of CNTFR

6.1.8. Indirect ¹²⁵ Goat Anti-Mouse Antibody Binding Assay

6.2. Results And Discussion

6.2.1. Restriction Analysis

6.2.2. In Vitro Transcription And Translation

6.2.3. Binding Analysis With CNTF

6.2.4. Sequence Of CNTFR And Homology To Other Growth Factor Receptors

7. Example:Tissue Localization Of Message For CNTFR

7.1. Materials And Methods

7.1.1. CNTFR Probe Preparation

7.1.2. RNA Preparation And Northern Blots

7.2. Results

8. Example: Evidence That The CNTF Receptor Is Linked To The CellSurface Via A Glycosyl-Phosphatidylinositol (GPI) Linkage

8.1. Materials and Methods

8.2. Results And Discussion

9. The Effects Of CNTF On Denervated Rat Skeletal Muscle In Vivo

9.1. The CNTF Receptor Is Expressed In Skeletal Muscle On Both MyotubesAnd Myoblasts

9.2. CNTF Prevents The Loss Of Both Muscle Weight And MyofibrillarProtein Content Associated With Denervation Atrophy

9.2.1. Denervation Surgery

9.2.2. Treatments

9.2.3. Muscle Weight And Protein Analysis

10. Deposit of Microorganisms

1. INTRODUCTION

The present invention relates to the ciliary neurotrophic factorreceptor (CNTFR), and provides for CNTF receptor encoding nucleic acidand amino acid sequences. It also relates to (i) assay systems fordetecting CNTF activity; (ii) experimental model systems for studyingthe physiological role of CNTF; (ii) diagnostic techniques foridentifying CNTF-related neurologic conditions; (iv) therapeutictechniques for the treatment of CNTF-related neurologic conditions, and(v) methods for identifying molecules homologous to CNTFR.

2. BACKGROUND OF THE INVENTION 2.1. Ciliary Neurotrophic Factor

Ciliary neurotrophic factor (CNTF) is a protein that is specificallyrequired for the survival of embryonic chick ciliary ganglion neurons invitro (Manthorpe et al., 1980, J. Neurochem. 34:69-75). The ciliaryganglion is anatomically located within the orbital cavity, lyingbetween the lateral rectus and the sheath of the optic nerve; itreceives parasympathetic nerve fibers from the oculomotor nerve whichinnervate the ciliary muscle and sphincter pupillae.

Ciliary ganglion neurons have been found to be among the neuronalpopulations which exhibit defined periods of cell death. In the chickciliary ganglion, half of the neurons present at embryonic day 8 (E8)have been observed to die before E14 (Landmesser and Pilar, 1974, J.Physiol. 241:737-749). During this same time period, ciliary ganglionneurons are forming connections with their target tissues, namely, theciliary body and the choriod coat of the eye. Landmesser and Pilar(1974, J. Physiol. 241:751-736) observed that removal of an eye prior tothe period of cell death results in the complete loss of ciliaryganglion neurons in the ipsilateral ganglion. Conversely, Narayanan andNarayanan (1978, J. Embryol. Ex. Morphol. 44:53-70) observed that, byimplanting an additional eye primordium and thereby increasing theamount of available target tissue, ciliary ganglion neuronal cell deathmay be decreased. These results are consistent with the existence of atarget derived neurotrophic factor which acts upon ciliary ganglionneurons.

In culture, ciliary ganglion (CG) neurons have been found to require afactor or factors for survival. Ciliary neurotrophic factor(s) (CNTF)activity has been identified in chick muscle cell conditioned media(Helfand et al., 1976, Dev. Biol. 50-541-547; Helfand et al., 1978, Exp.Cell Res. 113-39-45; Bennett and Nurcome, 1979, Brain Res. 173:543-548;Nishi and Berg, 1979, Nature 277-232-234; Varon et al., 1979, Brain Res.173:29-45), in muscle extracts (McLennan and Hendry, 1978, Neurosci.Lett. 10:269-273); in chick embryo extract (Varon et al., 1979, BrainRes. 173:29-45; Tuttle et al., 1980, Brain Res. 183:161-180), and inmedium conditioned by heart cells (for discussion, see also Adler etal., 1979, Science 204:1434-1436 and Barbin et al., 1984, J. Neurochem.43:1468-1478).

Adler et al. (1979, Science 204:1434-1436) used an assay system based onmicrowell cultures of CG neurons to demonstrate that a very rich sourceof CNTF was found in the intraocular target tissues the CG neuronsinnervate. Out of 8000 trophic units (TU) present in a twelve-dayembryo, 2500 TU were found present in eye tissue; activity appeared tobe localized in a fraction containing the ciliary body and choroid coat.

Subsequently, Barbin et al. (1984, J. Neurochem. 43:1468-1478) reporteda procedure for enriching CNTF from chick embryo eye tissue. CNTFactivity was also found to be associated with non-CG tissues, includingrat sciatic nerve (Williams et al., 1984, Int. J. Develop. Neurosci218:460-470). Manthorpe et al. (1986, Brain Res. 367:282-286) reportedpartial purification of mammalian CNTF activity from extracts of adultrat sciatic nerve using a fractionation procedure similar to thatemployed for isolating CNTF activity from chick eye. In addition,Watters and Hendry (1987, J. Neurochem. 49:705-713) described a methodfor enriching CNTF activity approximately 20,000-fold from bovinecardiac tissue under non-denaturing conditions using heparin-affinitychromatography. CNTF activity has also been identified in damaged braintissue (Manthorpe et al., 1983, Brain Res. 267:47-56; Nieto-Sampedro etal., 1983, J. Neurosci. 3:2219-2229).

Carnow et al. (1985, J. Neurosci. 5:1965-1971) and Rudge et al., (1987,Develop. Brain Res. 32:103-110) describe methods for identifyingCNTF-like activity from Western blots of tissue extracts and thenidentifying protein bands containing CNTF activity by inoculating thenitrocellulose strips in a culture dish with CG neurons and identifyingareas of cell survival using vital dyes. Using this method, Carnow etal. (1985, J. Neurosci. 5:1965-1971) observed that adult rat sciaticnerve and brain-derived CNTF activities appear to exhibit a differentsize (24 kD) than chick CNTF (20.4 kD).

Recently, CNTF has been cloned and synthesized in bacterial expressionsystems, as described in U.S. patent application Ser. No. 07/570,651,entitled "Ciliary Neurotrophic Factor," filed Aug. 20, 1990 by Sendtneret al. incorporated by reference in its entirety herein. Usingrecombinant probes, CNTF-mRNA in tissues of adult rat appeared to beabout 1.2 kb in size. Rat brain CNTF was cloned and found to be encodedby a mRNA having a short 5' untranslated region of 77 bp and an openreading frame of 600 bp, predicting a protein of about 200 amino acids(Stockli et al., 1989, Nature 342:920-923). Human CNTF was also clonedand sequenced (U.S. patent application Ser. No. 07/570,651, entitled"Ciliary Neurotrophic Factor," filed Aug. 20, 1990 by Sendtner et al.);its coding sequences were substantially conserved relative to ratsequences, whereas noncoding sequences were less conserved.

2.2. Functional Properties of Ciliary Neurotrophic Factor

A number of biological effects have been ascribed to CNTF. As discussedabove, CNTF was originally described as an activity which supported thesurvival of neurons of the E8 chick ciliary ganglion, a component of theparasympathetic nervous system. A description of other biologicalproperties of preparations known to contain CNTF activity follows:

Saadat et al. (1989, J. Cell Biol. 108:1807-1816) observed that theirmost highly purified preparation of rat sciatic nerve CNTF inducedcholinergic differentiation of rat sympathetic neurons in culture. Also,Hoffman (1988, J. Neurochem. 51:109-113) found that CNTF activityderived from chick eye increased the level ofcholine-O-acetyltransferase activity in retinal monolayer cultures.

Hughes et al. (1988, Nature 335:70-73) studied a population ofbipotential glial progenitor cells in cultures derived from theperinatal rat optic nerve and brain; these progenitor cells have beenshown to give rise to, first, oligodendrocytes and then, to type 2astrocytes. Under the culture conditions used, oligodendrocytedifferentiation appeared to occur directly from an oligodendrocyte-type2-astrocyte (0-2A) progenitor cell, whereas type 2 astrocytedifferentiation appears to require the presence of an inducing proteinsimilar or identical to CNTF (see also Anderson, 1989, Trends Neurosci.12:83-85).

Heymanns and Unsicker (1979, Proc. Natl. Acad. Sci. U.S.A. 4:7758-7762)observed that high-speed supernatants of neuroblastoma cell extractsproduced effects similar to those associated with CNTF activity fromchick eye or rat sciatic nerve; the presence of a protein similar butnot identical to CNTF (by molecular weight) was indicated.

Ebendal (1987, J. Neurosci. Res. 17:19-24) looked for CNTF-like activityin a variety of rat and chicken tissues. He observed CNTF-like activityamong a fairly wide range of rat, but not in chicken tissues; rat liver,spleen T cells, and submandibular gland cells were found to beassociated with low levels of CG survival promoting activity, whereasheart, brain, and skeletal muscle tissues were associated with highersurvival promoting activity. Among tissues tested the highest CNTF-likeactivity was observed to be associated with rat kidney.

While the above studies have shown that many tissue and cell extractscontain activities which support the survival of neuronal populationswhich are also responsive to CNTF, (i.e. they support the survival of E8chick ciliary ganglion neurons in a tissue culture bioassay), it cannotbe assumed that a single or identical protein is responsible for theseactivities. As shown for the family of fibroblast growth factors (FGFs)(Dionne et al., 1990, EMBO J. 9:2685-2692), for example, a number ofdistinct polypeptides or proteins possess identical biological activityin a single bioassay.

The neuronal specificity of chick eye and rat sciatic nerve CNTF wereinitially found to have some overlap with neuronal populationsresponsive to NGF. Although CNTF was observed to have some overlappingneuronal specificity with NGF, distinguishing characteristics betweenthem became most apparent in studies of the roles of CNTF and NGF inpopulations of developing neurons (Skaper and Varon, 1986, Brain Res.389:39-46). In addition to their differing roles in development, CNTFmay also be distinguished from NGF by molecular weight, isoelectricpoint, inability to be inactivated by antibodies to NGF, and by CNTF'sability to support the in vitro survival of CGF neurons (Barbin et al.,1984, J. Neurochem. 43:1468-1478). Lin et al. (1989), Science246:1023-1026 have reported that CNTF is without sequence homology toany previously reported proteins. Sendtner et al. (U.S. patentapplication Ser. No. 07/570,651, entitled "Ciliary Neurotrophic Factor,"filed Aug. 20, 1990) observed that recombinant CNTF promoted survival ofmediodorsal and ventral spinal cord neurons, and also that purified ratsciatic nerve CNTF appeared to prevent cell death of motorneurons inlesioned facial nerve (VIIth cranial nerve) of newborn rat (Sendtner etal., 1990, Nature 345:440-441).

The cloning and expression of CNTF using recombinant DNA technology hasled to the discovery of a number of CNTF activities.

2.3. Growth Factor Receptors

A number of receptors which mediate binding and response to proteinfactors have been characterized and molecularly clorecepter the last fewyears, including receptors for insulin, for platelet derived growthfactor, for epidermal growth factor and its relatives, for thefibroblast growth factors, and for various interleukins andhematopoietic growth factors. Recent data reveal that certain receptorscan bind to multiple (related) growth factors, while in other cases thesame factor can act on multiple (related) receptors (e.g. Lupu et al.,1990, Science 249:1552-1555; Dionne et al., 1990, EMBO J. 9:2685-2692;Miki et al., 1991, Science 251:72-75). Most receptors that bind proteinfactors can broadly be characterized as having extracellular portionsresponsible for specifically binding the factor, transmembrane regionswhich span the membrane, and intracellular domains that are ofteninvolved in initiating signal transduction upon binding of the proteinfactor to the receptor's extracellular portion. Interestingly, althoughmany receptors are comprised of a single polypeptide chain, otherreceptors apparently require (at least) two separate subunits in orderto bind to their factor with high-affinity and to allow functionalresponse following binding (e.g. Hempstead et al., 1989, Science243:373-375; Hibi et al., 1990, Cell 63:1149-1157). The extracellularand intracellular portions of a given receptor often share commonstructural motifs with the corresponding regions of other receptors,suggesting evolutionary and functional relationships between differentreceptors. These relationships can often be quite distant and may simplyreflect the repeated use of certain general domain structures. Forexample, a variety of different receptors that bind unrelated factorsmake use of "immunoglobulin" domains in their extracellular portions,while other receptors utilize "cytokine receptor" domains in theirfactor-binding regions (e.g. Akira et al., 1990, The FASEB J.4:2860-2867). A large number of receptors with distinct extracellularbinding domains (which thus bind different factors) contain relatedintracytoplasmic domains encoding tyrosine-specific protein kinases thatare activated in response to factor binding (e.g. Ullrich andSchlessinger, 1990, Cell 61:203-212). The mechanisms by whichfactor-binding "activates" the signal transduction process is poorlyunderstood, even in the case of receptor tyrosine kinases. For otherreceptors, in which the intracellular domain encodes a domain of unknownfunction or in which the binding component associates with a secondprotein of unknown function (e.g. Hibi et al., 1990, Cell 63:1149-1157),activation of signal transduction remains even more mysterious.

3. SUMMARY OF THE INVENTION

The present invention relates to CNTF receptor (CNTFR) genes andproteins. It is based, in part, on the cloning and characterization ofthe human CNTFR gene and its expression in transfected COS cells.

The present invention provides for nucleic acid sequences which encodethe CNTFR, as well as fragments derived therefrom. It also provides forsubstantially purified CNTFR protein, and for peptide fragments thereof.

In a further aspect of the invention, CNTFR probes, including nucleicacid as well as antibody probes, may be used to identify CNTFR-relatedmolecules. For example, the present invention provides for suchmolecules which form a complex with CNTFR and thereby participate inCNTFR function. As another example, the present invention provides forreceptor molecules which are homologous or cross-reactive antigenically,but not identical to CNTFR. These particular embodiments are based onthe discovery that the CNTFR bears homology to other biologicallyrelevant molecules, including, most particularly, the IL-6 receptor, butalso the PDGF receptor, the CSF-1 receptor, the prolactin receptor, theIL-2 and IL-4 receptors, the GM-CSF granulocyte macrophage colonystimulation factor receptor, pregnancy-specific alpha 1-betaglycoprotein, and carcinoembryonic antigen, a tumor marker.

The present invention also provides for assay systems for detecting CNTFactivity, comprising cells which express high levels of CNTFR, and whichare therefore extremely sensitive to even very low concentrations ofCNTF or CNTF-like molecules.

In addition, the present invention provides for experimental modelsystems for studying the physiological role of CNTF. Such systemsinclude animal models, such as (i) animals exposed to circulating CNTFRpeptides which compete with cellular receptor for CNTF binding andthereby produce a CNTF-depleted condition, (ii) animals immunized withCNTFR; (iii) transgenic animals which express high levels of CNTFR andtherefore are hypersensitive to CNTF; and (iv) animals derived usingembryonic stem cell technology in which the endogenous CNTFR genes weredeleted from the genome.

In yet further embodiments of the invention, CNTFR probes may be used toidentify cells and tissues which are responsive to CNTF in normal ordiseased states. For example, a patient suffering from a CNTF-relateddisorder may exhibit an aberrancy of CNTFR expression.

In addition, the CNTFR genes and proteins of the invention may be usedtherapeutically. For example, and not by way of limitation, acirculating CNTFR may be used to deplete CNTF levels in areas of traumato the central nervous system. Alternatively, a recombinant CNTFR genemay be inserted in tissues which would benefit from increasedsensitivity to CNTF, such as motorneurons in patients suffering fromamyotrophic lateral sclerosis.

4. DESCRIPTION OF THE FIGURES

FIGS. 1A to 1D2. FIG. 1A. Schematic diagram of expression cloning usingtagged ligand binding strategy. FIGS. 1B1 and 1B2. Secondary iodinatedantibody assay showed that in contrast to COS cells transfected with theoriginal cDNA library, many COS cells transfected with DNA obtainedafter one round of panning expressed CNTF-binding sites (radioautographdone on 60 mm plate of transfected COS cells; each black dot representsa single transfected COS cell expressing a CNTF-binding site). FIGS. 1C1and 1C2. The same assay as described in (FIGS. 1B1 and 1B2), but whereCOS cells had been transfected with a non-CNTFR encoding plasmid(negative clone) or a CNTFR encoding plasmid (positive clone).

Only small sections of each plate are shown. FIGS. 1D1 and 1D2. Resultsof fluorescence activated cell sorting (FACS) analysis of COS cellstransfected with the negative clone or the positive clone of FIGS. 1D1and 1D2.

FIGS. 2A to 2D. Nucleic acid sequence (SEQ ID NO:1) of CNTFR-encodingcDNA and deduced amino acid sequence (SEQ ID NO:2).

FIGS. 3A and 3D. Alignment of the human CNTFR showing homologies in theimmunoglobulin-like domain (FIG. 3A) and the cytokine receptor-likedomain (FIG. 3B). Numbers on the left indicate the amino acid numberstarting from the first methionine. Identical residues and conservedsubstitutions are marked by solid boxes. Gaps are introduced to maximizehomology. IL-6=interleukin 6 (IgG-like domain=SEQ ID NO:3, cytokine-likedomain=SEQ ID NO:8); CEA=carcinoembryonic antigen (IgG-like domain=SEQID NO:4), PDGF=platelet derived growth factor (IgG-like domain=SEQ IDNO:5), CSF-1=colony stimulating factor 1 (IgG-like domain=SEQ ID NO:6);alpha 1-βGP=alpha 1β glycoprotein (IgG-like domain=SEQ ID NO:7),PRL=prolactin (cytokine domain=SEQ ID NO:9), EPO=erythropoietin(cytokine domain=SEQ ID NO:10); IL-2=interleukin 2 (cytokine domain=SEQID NO:11); IL-4=interleukin 4 (cytokine domain=SEQ ID NO:12),GM-CSF=granulocyte macrophage colony stimulating factor (cytokinedomain=SEQ ID NO:13).

FIG. 4. Structural relationships between the CNTFR and other relatedreceptors. The human IL-6 receptor and CNTFR have an immunoglobulindomain fused to the N-terminus of the proposed factor binding domain. Ashort acidic tether (zig zag line) connects the globular immunoglobulinand proposed factor binding domain. A proposed protein similar to gp130is shown in association with the CNTFR, as discussed in the text.HuGRHR-human growth hormone receptor; RbPRLR-rabbit prolactin receptor;MOEPOR-mouse erythropoietin receptor; HuIL2Rβ-human interleukin-2receptor β-chain; HuIL6R-human interleukin 6 receptor; HuCNTFR-humanciliary derived neurotrophic factor receptor; C-cysteine; X- unknownamino acid; W-tryptophan; S-serine; F-phenylalanine.

FIG. 5. Tissue localization of CNTFR message. RNA was prepared from theindicated tissues of rat as described in section 8.1. DNA fragments ofCNTFR were derived from expression constructs containing these genes inpCMX as described in section 8.1. Tissues: cerebellum (CB); hindbrain(HB); midbrain (MB); thalamus (TH/HYP); striatum (STRI); hippocampus B(HIP B); hippocampus A (HIP A); cortex (CORT); olfactory bulb (OLF);adult brain (AD BR); skin (SK); heart (HRT); muscle (MUS), lung (LUNG);intestine (INT); kidney (KID); liver (LIV); spleen (SPL); thymus (THY);E17 liver (E17 LIV).

FIG. 6. pCMX with hCNTF-R gene insert. Construction of PCMX in copendingapplication.

FIG. 7. Northern blot analysis of CNTF receptor expression in skeletalmuscle. 10 μg of total RNA was run in each lane. Lane 1, mouse myoblastcell line C2C12 mb; lane 2, mouse myotube cell line C2C12 mt; lane 3,rat myotube cell line H9C2 mt; lane 4, rat myotube cell line L6 mt; lane5, rat soleus muscle; lane 6, rat extensor digitorum longus (EDL)muscle; lane 7, denervated skeletal muscle; lane 8, purified humanmyotubes; lane 9, skeletal muscle (this RNA sample was degraded); lane10, adult rat cerebellum; lane 11, sham-operated soleus muscle; lane 12,72 hour denervated rat soleus muscle; lane 13, sham-operated EDL muscle;lane 14, 72 hour denervated EDL muscle.

FIG. 8. Anatomical diagram of the right hindlimb subjected todenervation surgery.

FIG. 9. UNOP=unoperated soleus muscles from animal group 1 (Table IV);solid bar represents right side and stippled bar represents left side;NONE=lesioned (denervated, right side) and control (sham-operated, leftside) soleus muscles without any injection, from animal group 2;ALB=lesioned (right) and control (left) soleus muscles treated withPBS/BSA (SC) from animal group 6; CNTF=lesioned (right) and control(left) soleus muscles treated with CNTF/BSA (SC) from animal group 5.Solid bars: lesioned; stippled bars: control.

5. DETAILED DESCRIPTION OF THE INVENTION

For purposes of clarity of disclosure, and not by way of limitation, thedetailed description of the invention will be divided into the followingsubsections:

(i) cloning of the CNTF receptor;

(ii) nucleic acid encoding the CNTF receptor;

(iii) CNTFR peptides;

(iv) expression of CNTF receptor;

(v) identification of molecules related to the CNTF receptor; and

(vi) utility of the invention.

5.1. Cloning of the Ciliary Neurotrophic Factor Receptor

The present invention enables the cloning of the CNTF receptor (CNTFR)by providing a method for selecting target cells which express CNTFR. Byproviding a means of enriching for CNTFR encoding sequences, the presentinvention enables the purification of CNTFR protein and the directcloning of CNTFR-encoding DNA.

For example, CNTFR-bearing target cells may be selected, and CNTFRprotein may be purified using methods known to one skilled in the artfor the purification of a receptor molecule. For example, and not by wayof limitation, CNTF or CNTF attached to a detectable molecule, asdescribed in section 5.6.3, infra, in which the tag may be, for example,a radiolabel, antigenic determinant, or antibody, to name a few,(CNTF/tag) may be reversibly crosslinked to target cells, and membraneassociated proteins from said target cells may be subjected topurification methods. Such purification methods may include SDS-PAGE,followed by detection of the position of CNTF or CNTF/tag in the gel;for example, radiolabeled CNTF could be used, and, crosslinked to itsreceptor, may be visualized in the gel by autoradiography.Alternatively, anti-CNTF or anti-tag antibody could be used in theWestern blot technique to identify the position of the CNTF/receptorcomplex in such gels. Preparative gel electrophoresis could be used toisolate sufficient amounts of protein to enable amino acid sequencing ofpeptide fragments of the receptor, or to enable production of anti-CNTFRantibody which could be used to purify CNTFR molecules from target cellextracts. Amino acid sequence obtained from purified CNTFR may be usedto design degenerate oligonucleotide probes which may be used toidentify CNTFR encoding cloned nucleic acid in a genomic DNA library or,preferably, in a cDNA library constructed from CNTFR producing targetcells.

Alternatively, the CNTFR may be cloned by subtractive hybridizationmethods, in which mRNA may be prepared from target cells which expressCNTFR, and then non-CNTFR encoding sequences may be subtracted byhybridizing the MRNA (or cDNA produced therefrom) with mRNA or cDNAderived from cells such as neuronal cells which do not express theCNTFR. The nucleic acid remaining after subtraction is likely to beenriched in CNTFR-encoding sequences.

Nucleic acid prepared, preferably, from target cells enriched in CNTFRencoding sequences due to endogenous expression of CNTFR and/or due tosubtraction techniques discussed supra, may also be used in expressioncloning techniques to directly clone the CNTFR. For example, and not byway of limitation, total genomic DNA from target cells which expressCNTFR may be prepared and then transfected into a cell line which doesnot express CNTFR and which is preferably derived from a differentspecies from the target cell species (for example, DNA from a humanCNTFR-encoding cell may be transfected into a mouse cell, such as an Lcell). Although a relatively small number of transfected cells mayexpress CNTFR, such cells may be identified by resetting techniques orimmunofluorescence techniques as described in section 5.6.3, infra andmay be isolated, for example, by fluorescence-activated cell sorting orusing antibody-coupled magnetic beads or "panning" techniques, known toone skilled in the art. The CNTFR encoding DNA may be cloned fromreceptor-producing transfectants by producing a genomic library from thetransfectants and then isolating and propagating clones that containeither sequences conforming to CNTFR amino acid sequence or sequenceshomologous to species specific genetic elements; for example, human DNAmay be identified via Alu repeated sequences, which are distributed athigh frequency throughout the human genome. For example, and not by wayof limitation, cultured non-human cells comprising transfected human DNAencoding the CNTFR and which express human CNTFR may be selected,propagated, and then genomic DNA prepared from these cells may be usedto transfect cultured non-human cells, and CNTFR expressing cells may beselected. This process may be repeated; its purpose is to decrease, byeach transfection step, the amount of human DNA present in CNTFRencoding cells. Accordingly, when the genomic DNA of transfected, humanCNTFR expressing cells is cloned to generate a library, clones whichinclude human DNA (and are identified, for example, by screening fordistinctly human sequence elements) are more likely to compriseCNTFR-encoding sequences when repeated transfections have beenperformed.

RNA from a CNTFR expressing cell line or tissue source, or a cDNAexpression library obtained from such a source may be introduced inpools into Xenopus oocytes by direct injection; oocytes injected withpools encoding the CNTFR may be identified by assaying for functionalresponses (e.g. ion fluxes) that may be induced by exposing such oocytesto CNTF, or alternatively by detecting the presence of CNTF-bindingsites on the surface of such injected oocytes. Repetitively dividingpositive pools into smaller and smaller pools may lead to theidentification of individual clones encoding the CNTFR.

Alternatively, a cDNA expression library may be derived from CNTFRbearing target cells and then utilized in transient expression assays.In a preferred embodiment of the invention, said expression library mayincorporate the SV40 origin of replication and transient expressionassays may be performed using COS cells. CNTFR-expressing transfectantsmay be identified as set forth above, and CNTFR encoding DNA may beretrieved using standard methods. The nucleic acid sequence encoding theCNTFR may then be propagated and/or utilized in expression systems usingmethods substantially as set forth for nucleic acid encoding CNTF, asdescribed in U.S. patent application Ser. No. 07/570,651, entitled"Ciliary Neurotrophic Factor," filed Aug. 20, 1990 by Sendtner et al.

In a specific embodiment of the invention, exemplified in Section 6,infra, (and see FIG. 1A to 1D2) expression cloning of the CNTFR may beperformed as follows. A cDNA library may be prepared from a cell line ortissue which expresses CNTFR such as SH-SY5Y, such that the cDNA isinserted into an expression vector. This library may then be transfectedinto a suitable cell line, such as COS M5 cells, using, for example, aDEAE/chloroquine transfection protocol. Several days after transfection,the cells may be detached from their culture dishes and subjected to theAruffo/Seed panning procedure (Seed and Aruffo, 1987, Proc. Natl. Acad.Sci. U.S.A. 84:365-3369), with the following modifications:

(i) instead of incubating the transfected cells with anti-receptorantibodies, the cells may be incubated first with tagged CNTF (forexample, CNTF myc) on ice for about 30 minutes, centrifuged throughphosphate buffered saline (PBS)/2% Ficoll to remove excess ligand, andthen incubated with anti-tag antibody (for example, the anti-mycantibody 9E10) for about 30 minutes on ice.

(ii) the cells may then be spun through PBS/2% Ficoll and then "panned"on plates coated with antibody that recognizes the anti-tag antibody(for example, if the anti-tag antibody is 9E10, anti-mouse antibody.

Then, after washing nonadherent cells from the plates, Hirt supernatantsmay be prepared from the adherent cells, and plasmid DNA may beprecipitated in the presence of about 10-20 μg of tRNA. The resultingplasmid DNA may then be introduced into suitable bacteria (for exampleDH10 B bacteria) by standard techniques, including, but not limited to,electroporation. The cultures grown from transformed bacteria may thenbe used to prepare plasmid DNA for another round of eukaryotictransfection and panning. After this second transfection, panning andplasmid DNA preparation and transformation, the bacterial transformantsmay be plated out on selective media, individual colonies may be pickedand used for the preparation of plasmid DNA, and DNA prepared from anumber of such clones may be used individually for COS celltransfection. Alternatively, more rounds of enrichment may be necessarybefore individual colonies are tested. Resulting COS cells expressingCNTF binding sites may be identified by a number of techniques,including, but not limited to, indirect binding assays usingradioactively labeled or fluorescently labeled indicator antibodies. Anexample of a CNTFR-encoding nucleic acid is comprised in pCMX-hCNTFR(I2), FIG. 6, which has been deposited with the NRRL and assignedaccession number B-18789, and which is described in copending UnitedStates patent application Serial No. entitled "Mammalian ExpressionVector" by Davis and Yancopoulos. Clones identified in this manner maythen be analyzed by restriction fragment mapping and nucleic acidsequencing using standard techniques. Fragments of the CNTFR-encodingcDNA may then be used to identify genomic DNA sequences which comprisethe CNTFR gene, for example, from a genomic DNA library using standardhybridization techniques.

Once obtained, a CNTFR gene may be cloned or subcloned using any methodknown in the art. A large number of vector-host systems known in the artmay be used. Possible vectors include, but are not limited to, cosmids,plasmids or modified viruses, but the vector system must be compatiblewith the host cell used. Such vectors include, but are not limited to,bacteriophages such as lambda derivatives, or plasmids such as pBR322,pUC, or Bluescript® (Stratagene) plasmid derivatives. Recombinantmolecules can be introduced into host cells via transformation,transfection, infection, electroporation, etc.

The CNTFR gene may be inserted into a cloning vector which can be usedto transform, transfect, or infect appropriate host cells so that manycopies of the gene sequences are generated. This can be accomplished byligating the DNA fragment into a cloning vector which has complementarycohesive termini. However, if the complementary restriction sites usedto fragment the DNA are not present in the cloning vector, the ends ofthe DNA molecules may be enzymatically modified. It may proveadvantageous to incorporate restriction endonuclease cleavage sites intothe oligonucleotide primers used in polymerase chain reaction tofacilitate insertion into vectors. Alternatively, any site desired maybe produced by ligating nucleotide sequences (linkers) onto the DNAtermini; these ligated linkers may comprise specific chemicallysynthesized oligonucleotides encoding restriction endonucleaserecognition sequences. In an alternative method, the cleaved vector andCNTFR gene may be modified by homopolymeric tailing.

In specific embodiments, transformation of host cells with recombinantDNA molecules that incorporate an isolated CNTFR gene, cDNA, orsynthesized DNA sequence enables generation of multiple copies of thegene. Thus, the gene may be obtained in large quantities by growingtransformants, isolating the recombinant DNA molecules from thetransformants and, when necessary, retrieving the inserted gene from theisolated recombinant DNA.

5.2. Nucliec Acid Encoding Ciliary Neurotrophic Factor Receptor

Using the methods detailed supra and in Example Section 6, infra, thefollowing nucleic acid sequence was determined, and the correspondingamino acid sequence deduced. The sequence of the human CNTFR is depictedin FIGS. 2A to 2D (SEQ ID NO:1). This sequence, its functionalequivalent, or fragments of this sequence at least 6 nucleotides inlength may be used in accordance with the invention. Additionally, theinvention relates to CNTFR genes isolated from porcine, ovine, bovine,feline, avian, equine, or canine, as well as primate sources and anyother species in which CNTF activity exists. Subsequences comprisinghybridizable portions of the CNTFR sequence have use, e.g., in nucleicacid hybridization assays, Southern and Northern blot analyses, etc.

For example, the nucleic acid sequence depicted in FIGS. 2A to 2D (SEQID NO:1) can be altered by mutations such as substitutions, additions ordeletions that provide for sequences encoding functionally equivalentmolecules. According to the present invention, a molecule isfunctionally equivalent or active compared with a molecule having thesequence depicted in FIGS. 2A to 2D (SEQ ID NO:2) if it has the abilityto bind CNTF, but it does not necessarily bind CNTF with an affinitycomparable to that of natural CNTFR. Due to the degeneracy of nucleotidecoding sequences, other DNA sequences which encode substantially thesame amino acid sequence as depicted in FIGS. 2A to 2D (SEQ ID NO:2) maybe used in the practice of the present invention. These include but arenot limited to nucleotide sequences comprising all or portions of theCNTFR gene depicted in FIGS. 2A to 2D (SEQ ID NO:1) which is altered bythe substitution of different codons that encode a functionallyequivalent amino acid residue within the sequence, thus producing asilent change.

In addition, the recombinant CNTFR-encoding nucleic acid sequences ofthe invention may be engineered so as to modify processing or expressionof CNTFR. For example, and not by way of limitation, the CNTFR gene maybe combined with a promoter sequence and/or a ribosome binding site, ora signal sequence may be inserted upstream of CNTFR encoding sequencesto permit secretion of CNTFR and thereby facilitate harvesting orbioavailability.

Additionally, a given CNTFR can be mutated in vitro or in vivo, tocreate and/or destroy translation, initiation, and/or terminationsequences, or to create variations in coding regions and/or form newrestriction endonuclease sites or destroy preexisting ones, tofacilitate further in vitro modification. Any technique for mutagenesisknown in the art can be used, including but not limited to, in vitrosite-directed mutagenesis (Hutchinson, et al., 1978, J. Biol. Chem.253:6551), use of TAB® linkers (Pharmacia), etc.

5.3. Ciliary Neurotrophic Factor Receptor Peptides

The invention also provides for CNTFR proteins, fragments andderivatives thereof, having the amino acid sequence set forth in FIGS.2A to 2D (SEQ ID NO:2) or its functional equivalents and for proteinshomologous to such protein, such homology being of at least about 30percent. The invention also provides fragments or derivatives of CNTFRproteins which comprise at least six amino acids, comprise an antigenicdeterminant(s), or which are functionally active. The CNTFR proteinhaving the amino acid sequence depicted in FIGS. 2A to 2D (SEQ ID NO:2)has a molecular weight of approximately 42 kd.

CNTFR proteins, or fragments or derivatives thereof, of the inventioninclude, but are not limited to, those containing, as a primary aminoacid sequence, all or part of the amino acid sequence substantially asdepicted in FIGS. 2A to 2D (SEQ ID NO:2) including altered sequences inwhich functionally equivalent amino acid residues are substituted forresidues within the sequence resulting in a silent change. For example,one or more amino acid residues within the sequence can be substitutedby another amino acid of a similar polarity which acts as a functionalequivalent, resulting in a silent alteration. Substitutes for an aminoacid within the sequence may be selected from other members of the classto which the amino acid belongs. For example, the nonpolar (hydrophobic)amino acids include alanine, leucine, isoleucine, valine, proline,phenylalanine, tryptophan and methionine. The polar neutral amino acidsinclude glycine, serine, threonine, cysteine, tyrosine, asparagine, andglutamine. The positively charged (basic) amino acids include arginine,lysine and histidine. The negatively charged (acidic) amino acidsinclude aspartic acid and glutamic acid. Also included within the scopeof the invention are CNTFR proteins or fragments or derivatives thereofwhich are differentially modified during or after translation, e.g., byphosphorylation, glycosylation, crosslinking, acylation, proteolyticcleavage, linkage to an antibody molecule, membrane molecule or otherligand, (Ferguson et al., 1988, Ann. Rev. Biochem. 57:285-320).

The CNTFR peptides of the invention may be prepared by recombinantnucleic acid expression techniques or by chemical synthesis usingstandard peptide synthesis techniques.

5.4. Expression of Ciliary Neurotrophic Factor Receptor

In order to express recombinant CNTFR, the nucleotide sequence codingfor a CNTFR protein, or a portion thereof, can be inserted into anappropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedprotein-coding sequence. The necessary transcription and translationsignals can also be supplied by the native CNTFR gene and/or itsflanking regions. A variety of host-vector systems may be utilized toexpress the protein-coding sequence. These include but are not limitedto mammalian cell systems infected with virus (e.g., vaccinia virus,adenovirus, etc.); insect cell systems infected with virus (e.g.,baculovirus); microorganisms such as yeast containing yeast vectors, orbacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA.The expression elements of these vectors vary in their strengths andspecificities. Depending on the host-vector system utilized, any one ofa number of suitable transcription and translation elements may be used.

In a preferred specific embodiment of the invention, the CNTFR gene maybe comprised in the pCMX expression vector, as deposited with the NRRLand assigned accession no. B-18790.

Any of the methods previously described for the insertion of DNAfragments into a vector may be used to construct expression vectorscontaining a chimeric gene consisting of appropriatetranscriptional/translational control signals and the protein codingsequences. These methods may include in vitro recombinant DNA andsynthetic techniques and in vivo recombinations (genetic recombination).Expression of nucleic acid sequence encoding CNTFR protein or peptidefragment may be regulated by a second nucleic acid sequence so thatCNTFR protein or peptide is expressed in a host transformed with therecombinant DNA molecule. For example, expression of CNTFR may becontrolled by any promoter/enhancer element known in the art. Promoterswhich may be used to control CNTFR expression include, but are notlimited to, the SV40 early promoter region (Bernoist and Chambon, 1981,Nature 290:304-310), the CMV promoter, the promoter contained in the 3'long terminal repeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981,Proc. Natl. Acad. Sci. U.S.A. 78:144-1445), the regulatory sequences ofthe metallothionine gene (Brinster et al., 1982, Nature 296:39-42);prokaryotic expression vectors such as the β-lactamase promoter(Villa-Kamaroff, et al., 1978, Proc. Natl. Acad. Sci. U.S.A.75:3727-3731), or the tac promoter (DeBoer, et al., 1983, Proc. Natl.Acad. Sci. U.S.A. 80:21-25), see also "Useful proteins from recombinantbacteria" in Scientific American, 1980, 242:74-94; promoter elementsfrom yeast or other fungi such as the Gal 4 promoter, the ADC (alcoholdehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkalinephophatase promoter, and the following animal transcriptional controlregions, which exhibit tissue specificity and have been utilized intransgenic animals: elastase I gene control region which is active inpancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Ornitz etal., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald,1987, Hepatology 7:425-515); insulin gene control region which is activein pancreatic beta cells (Hanahan, 1985, Nature 315:115-122),immunoglobulin gene control region which is active in lymphoid cells(Grosschedl et al., 1984, Cell 38:647-658; Adames et al., 1985, Nature318:533-538; Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444),mouse mammary tumor virus control region which is active in testicular,breast, lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495),albumin gene control region which is active in liver (Pinkert et al.,1987, Genes and Devel. 1:268-276), alpha-fetoprotein gene control regionwhich is active in liver (Krumlauf et al., 1985, Mol. Cell. Biol.5:1639-1648; Hammer et al., 1987, Science 235:53-58); alpha1-antitrypsin gene control region which is active in the liver (Kelseyet al, 1987, Genes and Devel. 1:161-171), beta-globin gene controlregion which is active in myeloid cells (Mogram et al., 1985, Nature315:338-340; Kollias et al., 1986, Cell 46:89-94); myelin basic proteingene control region which is active in oligodendrocyte cells in thebrain (Readhead et al., 1987, Cell 48:703-712); myosin light chain-2gene control region which is active in skeletal muscle (Sani, 1985,Nature 314:283-286), and gonadotropic releasing hormone gene controlregion which is active in the hypothalamus (Mason et al., 1986, Science234:1372-1378).

Expression vectors containing CNTFR gene inserts can be identified bythree general approaches: (a) DNA-DNA hybridization, (b) presence orabsence of "marker" gene functions, and (c) expression of insertedsequences. In the first approach, the presence of a foreign geneinserted in an expression vector can be detected by DNA-DNAhybridization using probes comprising sequences that are homologous toan inserted CNTFR gene. In the second approach, the recombinantvector/host system can be identified and selected based upon thepresence or absence of certain "marker" gene functions (e.g., thymidinekinase activity, resistance to antibiotics, transformation phenotype,occlusion body formation in baculovirus, etc.) caused by the insertionof foreign genes in the vector. For example, if the CNTFR gene isinserted within the marker gene sequence of the vector, recombinantscontaining the CNTFR insert can be identified by the absence of themarker gene function. In the third approach, recombinant expressionvectors can be identified by assaying the foreign gene product expressedby the recombinant. Such assays can be based, for example, on thephysical or functional properties of the CNTFR gene product, forexample, by binding of the receptor to CNTF or to an antibody whichdirectly recognizes the CNTFR.

In an additional embodiment, cells which do not normally express CNTFRmay be transfected with recombinant-CNTFR encoding nucleic acid and thentested for the expression of functional CNTFR by exposing thetransfectants to CNTF and then testing for an increase in cAMP levels.

Once a particular recombinant DNA molecule is identified and isolated,several methods known in the art may be used to propagate it. Once asuitable host system and growth conditions are established, recombinantexpression vectors can be propagated and prepared in quantity. Aspreviously explained, the expression vectors which can be used include,but are not limited to, the following vectors or their derivatives:human or animal viruses such as vaccinia virus or adenovirus; insectviruses such as baculovirus; yeast vectors; bacteriophage vectors (e.g.,lambda), and plasmid and cosmid DNA vectors, to name but a few.

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Expression from certainpromoters can be elevated in the presence of certain inducers; thus,expression of the genetically engineered CNTFR protein may becontrolled. Furthermore, different host cells have characteristic andspecific mechanisms for the translational and post-translationalprocessing and modification (e.g., glycosylation, cleavage) of proteins.Appropriate cell lines or host systems can be chosen to ensure thedesired modification and processing of the foreign protein expressed.For example, expression in a bacterial system can be used to produce anunglycosylated core protein product. Expression in yeast may be used toproduce a glycosylated product. Expression in mammalian cells can beused to ensure "native" glycosylation of the heterologous CNTFR protein.Furthermore, different vector/host expression systems may effectprocessing reactions such as proteolytic cleavages to different extents.

Once a recombinant which expresses the CNTFR gene is identified, thegene product should be analyzed. This can be achieved by assays based onthe physical or functional properties of the product.

Once the CNTFR protein is identified, it may be isolated and purified bystandard methods including chromatography (e.g., ion exchange, affinity,and sizing column chromatography), centrifugation, differentialsolubility, or by any other standard technique for the purification ofproteins. In particular, CNTFR protein may be isolated by binding to anaffinity column comprising CNTF bound to a stationary support.

Nucleic acid sequences complementary to DNA or RNA sequences encodingCNTFR or a functionally active portion thereof are also provided. In aparticular aspect, antisense oligonucleotides can be synthesized, whichare complementary to at least a portion of CNTFR mRNA.

5.5. Identification of Molecules Related to the Ciliary NeurotrophicFactor Receptor

Multiple receptor-factor systems have been defined in which the samefactor can bind to multiple receptors (see supra). As this may be thecase for CNTF, the present invention allows for the identification ofany additional CNTF receptors by the identical scheme used to obtain theCNTFR described here, except for the source of RNA used to prepare thecDNA expression library. A source may be chosen that would be likely tobe expressing a distinct CNTF receptor; sources may be evaluated for thepresence of CNTF-binding not attributable to the CNTFR (genetic probesand antibody reagents generated from the CNTFR sequence may be used tocompare the protein responsible for CNTF binding in cell lines or tissuesources with the CNTF described here). In addition, because receptorsare known which bind to more than one related factor (see supra),identification of the CNTFR should allow identification of anyadditional native ligands which bind this receptor.

In a further aspect of the invention, the CNTFR sequence may be used inthe identification of CNTFR-related molecules. The CNTFR contains motifswhich are shared with a variety of other receptors. The extracellularportion of the CNTFR contains both an "immunoglobulin" domain at itsN-terminus, as well as a "cytokine receptor" domain which is separatedfrom the "immunoglobulin" domain by a short hinge region. Although manyreceptors have homology to either the "immunoglobulin" or "cytokinereceptor" domains, only one receptor- the IL-6 receptor- shares the sameparticular arrangement of these domains with the CNTFR. The IL-6receptor is thus the protein most related to the CNTFR. Interestingly,the IL-6 receptor is also similar to the CNTFR in that it has a veryshort intracytoplasmic domain which is apparently not required forinitiating responses upon IL-6 binding (Hibi et al., 1990, Cell63:1149-1157). Recently, a novel signal transducer for the IL-6receptor, termed gp 130, was molecularly cloned. This transducer doesnot bind IL-6 by itself, but it does confer high affinity binding to theIL-6 receptor and it is required to transduce the IL-6 signal (Hibi etal., 1990, Cell 63:1149-1157). Cloning of the CNTFR reveals that itshares important features with the IL-6 receptor that are not found inother known receptors, thus defining a new family of receptors.Homologies between these first two members of this receptor family, asdefined by the present invention, may be used to identify additionalrelated receptors by using DNA or antibody probes corresponding tohomologous regions, or by using a polymerase chain reaction strategytogether with degenerate oligonucleotides corresponding to sharedregions of amino acid homology (e.g. Maisonpierre et al., 1990, Science247:1146-1451). The present invention may also be used for the testingof whether the CNTFR utilizes the same signal transducer as the IL-6receptor, or whether it utilizes a related molecule. Finally, theidentification of CNTFR-related receptors should aid in theidentification of novel ligands that would bind to these receptors.

According to the present invention, by screening a DNA library(comprising genomic DNA or, preferably, cDNA) with oligonucleotidescorresponding to CNTFR sequence derived either from protein sequencedata or from the nucleic acid sequence set forth in FIGS. 2A to 2D (SEQID NO:1), clones may be identified which encode new members of thefamily described above. By decreasing the stringency of hybridization,the chances of identifying somewhat divergent members of the family maybe increased. It may also be desirable to use sequences substantiallyshared by members of the family which have been sequenced; such highlyconserved regions may be particularly useful in identifying additionalmembers of the family. Library screening may be performed using, forexample, the hybridization technique of Benton and Davis (1977, Science196:180) or Grunstein and Hogness (1975, Proc. Natl. Acad. Sci. U.S.A.72:3961-3965). Clones identified by hybridization may then be furtheranalyzed, and new family members may be identified by restrictionfragment mapping and sequencing techniques according to methods wellknown in the art.

It may be desirable to utilize polymerase chain reaction (PCR)technology (Saiki et al., 1985, Science 230:1350-1354) to identifyadditional members of the CNTFR superfamily. For example, sense andantisense primers corresponding to known CNTFR sequence may be used inPCR, preferably using cDNA as template. It may be desirable to designthese primers such that they include restriction enzyme cleavage siteswhich may facilitate the insertion of the products of PCR intoappropriate cloning vectors. The products of PCR may be inserted intosuitable vectors and the resulting clones may then be screened for newfamily members. Such screening may be performed using standardtechniques, including hybridization analysis using probes correspondingto known sequence. For example, a series of probes representingdifferent regions of a characterized CNTFR protein may be hybridized atlow stringency to duplicate filters carrying DNA from clones generatedusing PCR, as outlined above. It may be observed that various clones mayhybridize to some probes, but not others. New family members may also beidentified by increasing the stringency of the hybridization conditions,wherein new members not identical to probes derived from known memberswould hybridize less strongly at higher stringency. Alternatively, newfamily members may be identified by restriction mapping or sequencinganalysis using standard techniques to reveal differences in restrictionmaps or sequences relative to known family members.

In additional embodiments, the present invention provides for moleculeswhich form a complex with CNTFR and thereby may participate in CNTFRfunction. For example, it has been found that CNTFR does not, bysequence analysis, appear to possess a cytoplasmic domain; it may, infact, be joined to a membrane through GPI linkageglycosyl-phosphatidylinositol (reviewed in Ferguson et al., 1988, Ann.Rev. Biochem. 57:285-320). This suggests that at least one othermolecule forms an association with CNTFR to participate in signaltransduction across the cell membrane. Such a molecule may be, forexample, a protein such as GP130 that is found associated with IL-6R(Taga et al., 1989, Cell 58:573-581); this is particularly likely inlight of the homology between CNTFR and the IL-6 receptor. Moleculeswhich are associated with CNTFR at the cell membrane may be isolated andidentified by any method known in the art, including but not limited tochemical cross-linkage, coprecipitation with anti-CNTFR antibody, or viaa CNTF/tag, and/or by protein or lipid purification techniques.

Further, the present invention provides for molecules other than CNTFwhich may bind to CNTFR. Such molecules are defined as molecules whichcompete with CNTF, including other normal ligands, for CNTFR binding,and include peptides, peptide derivatives and non-peptide (e.g.peptidomimetic) compounds.

5.6. Utility of the Invention 5.6.1. Assay Systems

The present invention provides for assay systems in which CNTF activityor activities similar to CNTF activity resulting from exposure to apeptide or non-peptide compound may be detected by measuring aphysiological response to CNTF in a cell or cell line responsive to CNTFwhich expresses the CNTFR molecules of the invention. A physiologicalresponse may comprise any of the biological effects of CNTF, includingbut not limited to, those described in Section 2.2, supra, as well asthe transcriptional activation of certain nucleic acid sequences (e.g.promoter/enhancer elements as well as structural genes), CNTF-relatedprocessing, translation, or phosphorylation, the induction of secondaryprocesses in response to processes directly or indirectly induced byCNTF, and morphological changes, such as neurite sprouting, or theability to support the survival of cells such as ciliary ganglion cells,motorneurons, Purkinje cells, or hippocampal neurons, to name but a few.

In a preferred specific embodiment of the invention, the functionalinteraction between CNTF and the CNTFR may be observed by detecting anincrease in the production of "immediate early" primary response genesactivated in response to many growth factor-stimulated transmembranesignals, including, but not limited to, c-fos and c-jun. For example,the activation of immediate early genes may be detected by Northern blotanalysis of immediate early gene mRNA levels. In a preferred embodimentof the invention, c-fos or c-jun mRNA levels may be determined byNorthern blot analysis of mRNA prepared from target cells incubated withCNTF, wherein CNTF activity is evidenced by an increase in levels ofc-fos or c-jun. Of note, in particular embodiments of the invention,once target cells have been produced that contain recombinantCNTFR-encoding nucleic acid or selected by virtue of binding to CNTF, itmay be desirable to ensure that the target cells respondcharacteristically to CNTF or compounds with CNTF-like activity. In thecontext of the present invention, the term CNTF-like activity isconstrued to mean biological activity which is similar but may or maynot be identical to that of CNTF; such activities would include but arenot limited to those described in Section 2.2, supra or the activationof particular immediate early promoters such as the fos or junpromoters.

The present invention provides for the development of novel assaysystems which may be utilized in the screening of compounds for CNTF- orCNTF-like activity. Target cells which bind to CNTF may be produced bytransfection with CNTFR-encoding nucleic acid or may be identified andsegregated by, for example, fluorescent-activated cell sorting,sedimentation of rosettes, or limiting dilution as described in Section5.6.3, infra.

Once target cell lines are produced or identified, it may be desirableto select for cells which are exceptionally sensitive to CNTF. Suchtarget cells may bear a greater number of CNTFRs; target cells bearing arelative abundance of CNTFRs could be identified by selecting targetcells which bind to high levels of CNTF, for example cells which whenincubated with CNTF/tag and subjected to immunofluorescence assayproduce a relatively higher degree of fluorescence. Alternatively, cellswhich are exceptionally sensitive to CNTF may exhibit a relativelystrong biological response, such as a sharp increase in immediate earlygene products such as c-fos or c-jun, in response to CNTF binding. Bydeveloping assay systems using target cells which are extremelysensitive to CNTF, the present invention provides for methods ofscreening for CNTF or CNTF-like activity which are capable of detectinglow levels of CNTF activity.

In particular, using recombinant DNA techniques, the present inventionprovides for CNTF target cells which are engineered to be highlysensitive to CNTF. For example, the CNTF-receptor gene, cloned accordingto the methods set forth in Section 5.1, may be inserted into cellswhich are naturally CNTF responsive such that the recombinant CNTFR geneis expressed at high levels and the resulting engineered target cellsexpress a high number of CNTFRs on their cell surface.

Alternatively, or additionally, the target cells may be engineered tocomprise a recombinant gene which is expressed at high levels inresponse to CNTF/receptor binding. Such a recombinant gene maypreferably be associated with a readily detectable product. For example,and not by way of limitation, transcriptional control regions (i.e.promoter/enhancer regions) from an immediate early gene may be used tocontrol the expression of a reporter gene in a construct which may beintroduced into target cells. The immediate early gene/reporter geneconstruct, when expressed at high levels in target cells by virtue of astrong promoter/enhancer or high copy number, may be used to produce anamplified response to CNTFR binding. For example, and not by way oflimitation, a CNTF-responsive promoter (such as the c-fos or c-junpromoter) may be used to control the expression of detectable reportergenes including β-galactosidase, growth hormone, chloramphenicol acetyltransferase, neomycin phosphotransferase, luciferase, orβ-glucuronidase. Detection of the products of these reporter genes, wellknown to one skilled in the art, may serve as a sensitive indicator forCNTF or CNTF-like activity of pharmaceutical compounds.

The CNTFR-encoding or reporter gene constructs discussed above may beinserted into target cells using any method known in the art, includingbut not limited to transfection, electroporation, calcium phosphate/DEAEdextran methods, and cell gun, as well as the production of transgenicanimals bearing the above-mentioned constructs as transgenes, and fromwhich CNTF target cells may be selected using the methods discussedsupra.

Assay systems of the present invention enable the efficient screening ofpharmaceutical compounds for utility in the treatment of CNTF-associateddiseases. For example, and not by way of limitation, it may be desirableto screen a pharmaceutical agent for CNTF activity and therapeuticefficacy in cerebellar degeneration. In a specific embodiment of theinvention, Purkinje cells responsive to CNTF may be identified andisolated, and then cultured in microwells in a multiwell culture plate.Culture medium with added test agent, or added CNTF, in numerousdilutions may be added to the wells, together with suitable controls.The cells may then be examined for improved survival,neuritesprouting,and so forth, and the activity of test agent and CNTF, as wellas their relative activities, may be determined. As another example,motorneuron lesions have been shown to respond favorably to CNTF(Sendtner et al., 1990, Nature 345:440). It may, therefore, be desirableto identify CNTF-like compounds which can, like CNTF, preventmotorneuron cell death following axotomy. CNTF responsive motorneuronscould be utilized in assay systems to identify compounds useful intreating motorneuron diseases. Considering that CNTF has been found tobe effective in preventing motorneuron cell death following axotomy,which clearly is an extremely important observation when contemplatingtreatments for spinal cord injuries, amyotrophic lateral sclerosis, anddiabetic neuropathy, in designing drugs which would be effective intreating these disorders, including drugs which may be required to passthe blood brain barrier, it is essential to have access to a reliableand sensitive screening system such as the methods the present inventionprovide. For another example, if a particular disease is found to beassociated with a defective CNTF response in a particular tissue, arational treatment for the disease would be supplying the patient withexogenous CNTF. However, it may be desirable to develop molecules whichhave a longer half-life than endogenous CNTF, or which act as CNTFagonists, or which are targeted to a particular tissue. Accordingly, themethods of the invention can be used to produce efficient and sensitivescreening systems which can be used to identify molecules with thedesired properties. Similar assay systems could be used to identify CNTFantagonists.

5.6.2. Experimental Model Systems

The present invention also provides for experimental model systems forstudying the physiological role of CNTF. In these model systems, CNTFRprotein, peptide fragment, or a derivative thereof, may be eithersupplied to the system or produced within the system. Such model systemscould be used to study the effects of CNTF excess or CNTF depletion. Theexperimental model systems may be used to study the effects of increasedor decreased response to CNTF in cell or tissue cultures, in wholeanimals, in particular cells or tissues within whole animals or tissueculture systems, or over specified time intervals (including duringembryogenesis) in embodiments in which CNTFR expression is controlled byan inducible or developmentally regulated promoter. In particularembodiments of the invention, the CMV promoter may be used to controlexpression of CNTFR in transgenic animals. The term "transgenicanimals," as used herein, refers to non-human transgenic animals,including transgenic mosaics, which carry a transgene in some or all oftheir cells, which include any non-human species, and which are producedby any method known in the art, including, but not limited tomicroinjection, cell fusion, transfection, electroporation, etc. Forexample, the animals may be produced by a microinjection of zygotesmethod such as that set forth in "Brinster et al, 1989, Proc. Natl.Acad. Sci. U.S.A. 82:4438-4442.

The present invention also provides for model systems for autoimmunedisease in which an autoimmune response is directed toward CNTFR. Suchmodels comprise animals which have been immunized with immunogenicamounts of CNTFR and preferably found to produce anti-CNTFR antibodiesand/or cell-mediated immunity. To produce such a model system, it may bedesirable to administer the CNTFR in conjunction with an immuneadjuvant, such as Bacille Calmette Guerin (BCG).

5.6.2.1. Models for Increased CNTF Activity

For example, and not by way of limitation, an experimental model systemmay be created which may be used to study the effects of excess CNTFactivity. In such a system, the response to CNTF may be increased byengineering an increased number of CNTFRs on cells of the model systemrelative to cells which have not been so engineered. It may bepreferable to provide an increased number of CNTFRs selectively on cellswhich normally express CNTFRs.

Cells may be engineered to produce increased numbers of CNTFR byinfection with a virus which carries a CNTFR gene of the invention.Alternatively, the CNTFR gene may be provided to the cells bytransfection.

If the model system is an animal, a recombinant CNTFR gene may beintroduced into the cells of the animal by infection with a virus whichcarries the CNTFR gene. Alternatively, a transgenic animal may becreated which carries the CNTFR gene as a transgene.

In order to ensure expression of CNTFR, the CNTFR gene should be placedunder the control of a suitable promoter sequence. It may be desirableto put the CNTFR gene under the control of a constitutive and/or tissuespecific promoter, including but not limited to the CNS neuron specificenolase, neurofilament, and tyrosine hydroxylase promoter, an induciblepromoter, such as the metallothionein promoter, the UV activatedpromoter in the human immunodeficiency virus long terminal repeat(Valeri et al., 1988, Nature 333:78-81), or the CMV promoter (ascontained in PCMX, infra) or a developmentally regulated promoter.

By increasing the number of cellular CNTFRs, the response to endogenousCNTF may be increased. If the model system contains little or no CNTF,CNTF may be added to the system. It may also be desirable to addadditional CNTF to the model system in order to evaluate the effects ofexcess CNTF activity. Over expressing CNTF (or secreted CNTF) may be thepreferable method for studying the effects of elevated levels of CNTF oncells already expressing CNTFR. More preferably would be to expressCNTFR in all cells (general expression) and determine which cells arethen endowed with functional responsiveness to CNTF, thus allowing thepotential identification of a second receptor component, if one exists.

5.6.2.2. Models for Decreased CNTF Activity

Alternatively, as an example, and not by way of limitation, anexperimental model system may be created which may be used to study theeffects of diminished CNTF activity. This system may permitidentification of processes or neurons which require CNTF, and which mayrepresent potential therapeutic targets. In such a system, the responseto CNTF may be decreased by providing recombinant CNTFRs which are notassociated with a cell surface or which are engineered so as to beineffective in transducing a response to CNTF.

For example, CNTFR protein, peptide, or derivative may be supplied tothe system such that the supplied receptor may compete with endogenousCNTFR for CNTF binding, thereby diminishing the response to CNTF. TheCNTFR may be a cell free receptor which is either added to the system orproduced by the system. For example, a CNTFR protein which lacks thetransmembrane domain may be produced by cells within the system, such asan anchorless CNTFR that may be secreted from the producing cell.Alternatively, CNTFR protein, peptide or derivative may be added to anextracellular space within the system.

In additional embodiments of the invention, a recombinant CNTFR gene maybe used to inactivate or "knock out" the endogenous gene by homologousrecombination, and thereby create a CNTFR deficient cell, tissue, oranimal. For example, and not by way of limitation, a recombinant CNTFRgene may be engineered to contain an insertional mutation, for examplethe neo gene, which inactivates CNTFR. Such a construct, under thecontrol of a suitable promoter, may be introduced into a cell, such asan embryonic stem cell, by a technique such as transfection,transduction, injection, etc. Cells containing the construct may then beselected by G418 resistance. Cells which lack an intact CNTFR gene maythen be identified, e.g. by Southern blotting or Northern blotting orassay of expression. Cells lacking an intact CNTFR gene may then befused to early embryo cells to generate transgenic animals deficient inCNTFR. A comparison of such an animal with an animal not expressingendogenous CNTF would reveal that either the two phenotypes matchcompletely or that they do not, implying the presence of additionalCNTF-like factors or receptors.

Such an animal may be used to define specific neuronal populations, orany other in vivo processes, normally dependent upon CNTF. Thus, thesepopulations or processes may be expected to be effected if the animaldid not express CNTFR and therefore could not respond to CNTF.

Alternatively, a recombinant CNTFR protein, peptide, or derivative whichcompetes with endogenous receptor for CNTF may be expressed on thesurface of cells within the system, but may be engineered so as to failto transduce a response to CNTF binding.

The recombinant CNTFR proteins, peptides or derivatives described abovemay bind to CNTF with an affinity that is similar to or different fromthe affinity of endogenous CNTFR to CNTF. To more effectively diminishthe response to CNTF, the CNTFR protein, peptide, or derivative maydesirably bind to CNTF with a greater affinity than that exhibited bythe native receptor.

If the CNTFR protein, peptide, or derivative is produced within themodel system, nucleic acid encoding the CNTFR protein, peptide, orderivative may be supplied to the system by infection, transduction,transfection, etc. or as a transgene. As discussed supra, the CNTFR genemay be placed under the control of a suitable promoter, which may be,for example, a tissue-specific promoter or an inducible promoter ordevelopmentally regulated promoter.

In a specific embodiment of the invention the endogenous CNTFR gene of acell may be replaced by a mutant CNTFR gene by homologous recombination.

In a further embodiment of the invention, CNTFR expression may bereduced by providing CNTFR expressing cells with an amount of CNTFRanti-sense RNA or DNA effective to reduce expression of CNTFR protein.

5.6.3. Diagnositic Applications

According to the present invention, CNTFR probes may be used to identifycells and tissues which are responsive to CNTF in normal or diseasedstates. The present invention provides for methods for identifying cellswhich are responsive to CNTF comprising detecting CNTFR expression insuch cells. CNTFR expression may be evidenced by transcription of CNTFRmRNA or production of CNTFR protein. CNTFR expression may be detectedusing probes which identify CNTFR nucleic acid or protein.

One variety of probe which may be used to detect CNTFR expression is anucleic acid probe, which may be used to detect CNTFR-encoding RNA byany method known in the art, including, but not limited to, in situhybridization, Northern blot analysis, or PCR related techniques.

Another variety of probe which may be used is tagged CNTF, as set forthin U.S. Ser. No. 07/532,285now abandoned, the complete text of which isincorporated by reference herein.

According to the present invention, the term "tagged" CNTF should beconstrued to mean a CNTF molecule which is attached to a seconddetectable compound (the "tag"). The detectable compound may compriseradioisotope, a fluorescent moiety, or a ligand capable of binding to areceptor, or a substance which may be detected colorimetrically or whichhas catalytic activity. In preferred embodiments, the tag may comprisean antigenic determinant such that antibody is capable of binding to thetag. In alternative embodiments the tag itself may be an antibody; in aspecific embodiment of the invention the tag is monoclonal antibodyRP3-17. It is desirable that the tag not interfere with the biologicalactivity of CNTF and that the methods of detection of the tag would notsubstantially interfere with the binding of CNTF to its receptor.

The tag may be attached to CNTF using any method known in the art. Inpreferred embodiments of the invention, the tag is covalently linked toCNTF but in some cases it may be desirable that the tag be attached bynoncovalent forces (for example, if the tag comprises an immunoglobulinmolecule).

The tag may be of any molecular size suitable for preserving itsdetector function without substantially altering the biological activityof the attached CNTF. If the tag is to provide an antigenic determinant,it may be desirable that it comprise at least about 5-15 amino acids.

For purposes of illustration, and not by way of limitation, in onepreferred specific method of the invention, CNTF may be tagged using a"patch" polymerase chain reaction in which recombinant neurotrophicfactor (CNTF) is engineered to carry at its C-terminal end ten aminoacids corresponding to a known antigenic determinant. For example, andnot by way of limitation, this antigenic determinant may correspond to adefined epitope of the human c-myc proto-oncogene protein.

For example, and not by way of limitation, the "patch" PCR method may beused to attach the ten amino acid myc tag as follows (the presentinvention provides for any amino acid tag attached by analogousmethods). A 5' PCR primer corresponding to an CNTF sequence upstream ofa unique restriction enzyme cleavage site in a bacterial expressionconstruct may be utilized in PCR reaction with a "patch" primercomprising nucleic acid sequence corresponding to 3' terminal CNTFsequence and nucleic acid sequence encoding the peptide tag, using cDNAfrom CNTF-responsive cells as template.

The PCR reaction should also comprise a 3' primer corresponding to thepatch primer sequence and including nucleic acid sequence whichincorporates unique restriction endonuclease cleavage sites. Inpreferred embodiments, the 5' and 3' primers may be used in excess ofpatch primer, such that PCR amplification between 5' and patch primersmay cease after a few PCR cycles whereas amplification between the 5'and 3' primers may initiate and continue to produce a high yield of fulllength CNTF/tag sequence. The "patch" technique overcomes the need forlong primers whose synthesis may be difficult and time consuming. Theamplified CNTF/tag product may be gel purified, digested withrestriction enzymes which cleave at the sites engineered into thetermini of the product, and then subcloned into the correspondingrestriction sites of an expression vector. For example, to produceCNTF-myc tag, the following primers may be used: 5' primer=5' GAC TCGAGT CGA CAT CGG AGG CTG ATG GGA TGCC 3' (SEQ ID NO:14); patch primer=3'CTA AAG ACT CCT CCT AGA CAT CGC CGG CGT ATCG 5' (SEQ ID NO:15); primersmay be used in a ratio of 100 ng 5' primer/100 ng 3' primer/1 ng patchprimer; for details see Section 6, infra. The expression of CNTF/tag maybe carried out as described for the expression of recombinant CNTF inU.S. patient application Ser. No. 07/570,651, entitled "CiliaryNeurotrophic Factor," filed Aug. 20, 1990 or PCT Publication NoW091/04316, published Apr. 4, 1991 by Sendtner et al.

The present invention also provides for a tag which comprises animmunoglobulin molecule, or a portion thereof, e.g. an Fc, F(ab)₂, orF(ab)' fragment of an antibody molecule. The tag should bind to CNTF,and may be a polyclonal or monoclonal antibody.

According to the invention, tagged CNTF may be incubated with cellsunder conditions which would promote the binding or attachment of CNTFto said cells. In most cases, this may be achieved under standardculture conditions. For example, in a preferred embodiment of theinvention, cells may be incubated for about 30 minutes in the presenceof tagged CNTF. If the tag is an antibody molecule, it may be preferableto allow CNTF to bind to cells first and subsequently wash cells toremove unbound ligand and then add anti-CNTF antibody tag.

In particular embodiments of the invention, tagged CNTF on the surfaceof CNTF-responsive cells, hereafter called target cells, may be detectedby rosetting assays in which indicator cells that are capable of bindingto the tag are incubated with cells bearing CNTF/tag such that theyadhere to CNTF/tag on the target cells and the bound indicator cellsform rosette-like clusters around CNTF-tag bearing cells. These rosettesmay be visualized by standard microscopic techniques on plated cells,or, alternatively, may allow separation of rosetted and non-rosettedcells by density centrifugation. In a preferred specific embodiment ofthe invention, target cells, such as neuronal cells, may be harvestedand plated at a concentration of about 200 cells/well in a multiple well(e.g. 60 well) culture plate in medium such as RPM1 1640 with 10% fetalbovine serum and 2 mM glutamine. Plated cells may be incubated for about16 to 24 hours at 37° C. in a humidified 5% CO₂ atmosphere incubator toallow cells to attach. Next, excess cell culture media may be removedand the cells may be incubated for about 30 minutes at room temperaturewith tagged CNTF. The cells may then be washed several times with PBS(with calcium and magnesium) supplemented with 1% bovine serum albumin(BSA) to remove unbound ligand and then incubated for about 30 minutesat room temperature with about 10 μg/ml of antibody which recognizes thetag molecule. Cells may then be washed several times with PBS to removeunbound antibody. Then, the target cells (bearing CNTF/tag bound toanti-tag antibody) may be incubated at room temperature for 1 hour withabout a 0.2% (v/v) suspension of resetting indicator cells which bind tothe anti-tag antibody (such as indicator cells bearing rabbit-anti-mouseimmunoglobulin). The plates may then be washed with PBS and examinedunder a phase contrast microscope for rosettes. For example, if theanti-tag antibody is produced by a mouse, indicator cells may beproduced by coating erythrocytes (such as human o+erythrocytes) withanti-(mouse immunoglobulin) antibody produced by another species.Indicator cells may be prepared by incubating erythrocytes withanti-immunoglobulin antibody (at a concentration greater than about 1mg/ml) in the presence of 0.01% CrCl₃. 6H₂ O diluted in saline accordingto the procedure of Albino et al. (1981, J. Exp. Med. 154:1764-1778).Alternatively, magnetic beads or other methods known in the art may beused.

In alternative embodiments of the invention, tagged CNTF on the surfaceof target cells may be detected using immunofluorescent techniques inwhich a molecule which reacts with the tag, preferably an antibody,directly or indirectly produces fluorescent light. The fluorescence mayeither be observed under a microscope or used to segregateCNTF/tag-bearing cells by fluorescence activated cell sortingtechniques. In a preferred specific embodiment of the inventionpresented by way of example, target cells may be triturated andresuspended in assay buffer containing CNTF/tag (in excessconcentration) and sodium azide (0.05%) for about 30 minutes at 4° C.Cells may then be washed three times in assay buffer by centrifugationat 800 rpm for 5 minutes. Cells may then be incubated with anti-tagantibody at a concentration of about 10 μg/ml for about 30 minutes at 4°C., washed as above, and then incubated for about 30 minutes at 4° C.with biotinylated anti-immunoglobulin and streptavidin-Texas Redconjugate. The cells may then be washed, resuspended in mountingsolution, coverslipped, and then examined by fluorescent microscopy.

The present invention also provides for methods for detecting otherforms of tags, such as chromogenic tags, catalytic tags, etc. Thedetection methods for any particular tag will depend on the conditionsnecessary for producing a signal from the tag, but should be readilydiscernible by one skilled in the art.

Yet another variety of probe which may be used is anti-CNTFR antibody.

According to the invention, CNTFR protein, or fragments or derivativesthereof, may be used as an immunogen to generate anti-CNTFR antibodies.By providing for the production of relatively abundant amounts of CNTFRprotein using recombinant techniques for protein synthesis (based uponthe CNTFR nucleic acid sequences of the invention), the problem oflimited quantities of CNTFR has been obviated.

To further improve the likelihood of producing an anti-CNTFR immuneresponse, the amino acid sequence of CNTFR may be analyzed in order toidentify portions of the molecule which may be associated with increasedimmunogenicity. For example, the amino acid sequence may be subjected tocomputer analysis to identify surface epitopes which presentcomputer-generated plots of hydrophilicity, surface probability,flexibility, antigenic index, amphiphilic helix, amphiphilic sheet, andsecondary structure of CNTFR. Alternatively, the deduced amino acidsequences of CNTFR from different species could be compared, andrelatively non-homologous regions identified; these non-homologousregions would be more likely to be immunogenic across various species.

For preparation of monoclonal antibodies directed toward CNTFR, anytechnique which provides for the production of antibody molecules bycontinuous cell lines in culture may be used. For example, the hybridomatechnique originally developed by Kohler and Milstein (1975, Nature256:495-497), as well as the trioma technique, the human B-cellhybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., 1985, in "Monoclonal Antibodies and Cancer Therapy," Alan R.Liss, Inc. pp. 77-96) and the like are within the scope of the presentinvention.

The monoclonal antibodies for therapeutic use may be human monoclonalantibodies or chimeric human-mouse (or other species) monoclonalantibodies. Human monoclonal antibodies may be made by any of numeroustechniques known in the art (e.g., Teng et al., 1983, Proc. Natl. Acad.Sci. U.S.A. 80:7308-7312; Kozbor et al., 1983, Immunology Today 4:72-79;Olsson et al., 1982, Meth. Enzymol. 92:3-16). Chimeric antibodymolecules may be prepared containing a mouse antigen-binding domain withhuman constant regions (Morrison et al., 1984, Proc. Natl. Acad. Sci.U.S.A. 81:6851, Takeda et al., 1985, Nature 314:452).

Various procedures known in the art may be used for the production ofpolyclonal antibodies to epitopes of CNTFR. For the production ofantibody, various host animals can be immunized by injection with CNTFRprotein, or fragment or derivative thereof, including but not limited torabbits, mice, rats, etc. Various adjuvants may be used to increase theimmunological response, depending on the host species, and including butnot limited to Freund's (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful human adjuvants suchas BCG (Bacille Calmette-Guerin) and, Corynebacterium parvum.

A molecular clone of an antibody to a CNTFR epitope can be prepared byknown techniques. Recombinant DNA methodology (see e.g., Maniatis etal., 1982, Molecular Cloning, A Laboratory Manual, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.) may be used to construct nucleicacid sequences which encode a monoclonal antibody molecule, or antigenbinding region thereof.

Antibody molecules may be purified by known techniques, e.g.,immunoabsorption or immunoaffinity chromatography, chromatographicmethods such as HPLC (high performance liquid chromatography), or acombination thereof, etc.

The present invention provides for antibody molecules as well asfragments of such antibody molecules. Antibody fragments which containthe idiotype of the molecule can be generated by known techniques. Forexample, such fragments include but are not limited to: the F(ab')₂fragment which can be produced by pepsin digestion of the antibodymolecule; the Fab' fragments which can be generated by reducing thedisulfide bridges of the F(ab')₂ fragment, and the Fab fragments whichcan be generated by treating the antibody molecule with papain and areducing agent.

The abovementioned probes may be used experimentally to identify cellsor tissues which hitherto had not been shown to express CNTFR.Furthermore, these methods may be used to identify the expression ofCNTFR by aberrant tissues, such as malignancies. In additionalembodiments, these methods may be used diagnostically to compare theexpression of CNTFR in cells, fluids, or tissue from a patient sufferingfrom a disorder with comparable cells, fluid, or tissue from a healthyperson. Fluid is construed to refer to any body fluid, but particularlyblood or cerebrospinal fluid. A difference in the levels of expressionof CNTFR in the patient compared to a healthy person may indicate thatthe patient's disorder may be primarily or secondarily related to CNTFmetabolism. An increase in levels of CNTFR, for example, could eitherindicate that the patient's disorder is associated with an increasedsensitivity to normal levels of CNTF or, alternatively, may suggest thatthe patient's CNTF levels are low such that the number of receptors isincreased by way of compensation. These etiologies may be distinguishedfrom one another by administering CNTF to the patient. If his conditionworsens, he may suffer from CNTF hypersensitivity; if it improves, hemay be suffering from a CNTF deficiency. CNTF or CNTF antagonist-basedtherapeutic regimens may be chosen accordingly. Differences inexpression can be detected at the protein and/or RNA level; i.e. bymeasuring amounts of CNTFR protein or CNTFR RNA in a patient relative tothose amounts in healthy persons.

The abovementioned probes may also be used to select CNTF-responsivecells for use in assay systems, as described above, or in U.S.application Ser. No. 07/532,285, now abandoned, or according to standardmethods of cell selection or cell sorting.

5.6.4. Therapeutic Applications

The present invention also provides for methods in which a patientsuffering from a disorder, such as neurologic disorder is treated withan effective amount of CNTFR protein, peptide fragment, or derivative ofthe invention. Therapeutic methods comprising administering CNTFR, CNTFRagonists, CNTFR antagonists (which compete with endogenous CNTF), oranti-CNTFR antibodies are within the scope of the present invention.

The present invention also provides for pharmaceutical compositionscomprising CNTFR protein, peptide fragment, or derivative in a suitablepharmacologic carrier.

The CNTFR protein, peptide fragment, or derivative may be administeredsystemically or locally. Any appropriate mode of administration known inthe art may be used, including, but not limited to, intravenous,intrathecal, intraarterial, intranasal, oral, subcutaneous,intraperitoneal, or by local injection or surgical implant. sustainedrelease formulations are also provided for.

As our understanding of neurodegenerative disease/neurotrauma becomesclearer, it may become apparent that it would be beneficial to decreasethe trophic effect of endogenous CNTF. Therefore, in areas of nervoussystem trauma, it may be desirable to provide CNTF antagonists,including, but not limited to, cell-free CNTFR which may compete withendogenous cellular receptor for CNTF binding. Under such circumstances,it may be desirable to provide CNTF antagonist locally at the injurysite rather than systemically. Use of a CNTFR providing implant may bedesirable.

Alternatively, certain conditions may benefit from an increase in CNTFresponsiveness. It may therefore be beneficial to increase the number orbinding affinity of CNTFRs in patients suffering from such conditions.This could be achieved through gene therapy. Selective expression ofrecombinant CNTFR in appropriate cells could be achieved using CNTFRgenes controlled by tissue specific or inducible promoters or byproducing localized infection with replication defective virusescarrying a recombinant CNTFR gene. Conditions which may benefit fromincreased sensitivity to CNTF include particularly but are not limitedto motorneuron disorders including amyotrophic lateral sclerosis,Werdnig-Hoffmann disease, chronic proximal spinal muscular atrophy, andpost-polio syndrome. Such treatment may also be used for treatment ofneurological disorders associated with diabetes, Parkinson's disease,Alzheimer's disease, and Huntington's chorea.

Further, the invention provides for treatment of disorders of a specifictissue or cell-type by administration of CNTF, which tissue or cell-typehas been identified as expressing CNTF receptors. In a specificembodiment, it has been shown that the CNTFR gene is expressed in musclecells (see Section 8, infra), and that CNTF prevents the loss of bothmuscle weight and myofibrillar protein content associated withdenervation atrophy in vivo (see Section 9, infra). Accordingly, thepresent invention provides for methods of treating muscle celldisorders, or disorders involving the neuromuscular unit, comprisingadministering to a patient in need of such treatment (i) a nucleic acidmolecule comprising a nucleotide sequence which encodes CNTFR or afunctionally active portion or derivative thereof, such that it can beexpressed, or (ii) CNTF, or a functionally active portion or derivativethereof. Such disorders include but are not limited to those in whichatrophic or dystrophic change of muscle is the fundamental pathologicalfinding. For example, such muscle atrophy can result from denervation(loss of contact by the muscle with its nerve) due to nerve trauma;degenerative, metabolic, or inflammatory (e.g., Guillian-Barre syndrome)peripheral neuropathy, or damage to nerves caused by environmentaltoxins or drugs. In another embodiment, the muscle atrophy results fromdenervation due to a motor neuronopathy. Such motor neuronopathiesinclude, but are not limited to: adult motor neuron disease, includingAmyotrophic Lateral Sclerosis (ALS or Lou Gehrig's disease); infantileand juvenile spinal muscular atrophies, and autoimmune motorneuronopathy with multifocal conduction block. In another embodiment,the muscle atrophy results from chronic disuse. Such disuse atrophy maystem from conditions including, but not limited to: paralysis due tostroke, spinal cord injury, brain trauma or other Central Nervous Systeminjury; skeletal immobilization due to trauma (such as fracture, sprainor dislocation) or prolonged bed rest. In yet another embodiment, themuscle atrophy results from metabolic stress or nutritionalinsufficiency, including, but not limited to, the cachexia of cancer andother chronic illnesses, fasting or rhabdomyolysis, endocrine disorderssuch as, but not limited to, disorders of the thyroid gland anddiabetes. The muscle atrophy can also be due to a muscular dystrophysyndrome, including but not limited to the Duchenne, Becker, myotonic,Fascioscapulohumeral, Emery-Dreifuss, oculopharyngeal, scapulohumeral,limb girdle, and congenital types, and the dystrophy known as HereditaryDistal Myopathy. In a further embodiment, the muscle atrophy is due to acongenital myopathy, including, but not limited to Benign CongenitalHypotonia, Central Core disease, Nemaline Myopathy, and Myotubular(centronuclear) myopathy. In addition, CNTFR-encoding nucleic acids orCNTF and its active fragments or derivatives may be of use in thetreatment of acquired (toxic or inflammatory) myopathies. Myopathieswhich occur as a consequence of an inflammatory disease of muscle,include, but are not limited to polymyositis and dermatomyositis. Toxicmyopathies may be due to agents including, but not limited toamiodarone, chloroquine, clofibrate, colchicine, doxorubicin, ethanol,hydroxychloroquine, organophosphates, perihexiline, and vincristine.

In a further embodiment of the invention, patients that suffer from anexcess of CNTFR, hypersensitivity to CNTF, excess CNTF, etc. may betreated by administering an effective amount of anti-sense RNA oranti-sense oligodeoxyribonucleotides corresponding to the CNTFR genecoding region thereby decreasing expression of CNTFR.

6. EXAMPLE: EXPRESSION CLONING OF THE CILIARY NEUROTROPHIC FACTORRECEPTOR 6.1. Materials And Methods 6.1.1. Construction of aCNTF-receptor Expression Library

SH-SY5Y cells (originally obtained from Dr. June Biedler) were used as asource of mRNA for construction of a cDNA library using the pCMXexpression vector (described in copending U.S. patent application Ser.No. 07/678,408 filed Mar. 28, 1991, see supra), a derivative of thepCDM8 vector (Seed, 1987, Nature 329:840-842). Inserts for the cDNAlibrary were selected on an agarose gel for sizes larger than 1 kb.

6.1.2. "Panning" Method

The "panning" method developed by Seed and Aruffo (1987, Proc. Natl.Acad. Sci. U.S.A. 84:3365-3369) was modified as follows: instead ofincubating the cells with antibodies recognizing the receptor, cellswere incubated first with CNTF/myc (1 μg/ml) on ice for 30 minutes, spunthrough PBS/2% Ficoll to remove excess ligand, and then incubated with9E10 antibody obtained from Oncogene Sciences, Manhasset, N.Y. for 30minutes on ice. This was followed by another spin through PBS/2% Ficolland "panning" on plates coated with anti-myc peptide mouse monoclonalantibody obtained from Sigma. The plates were prepared as follows:bacteriological 60 mm plates (Falcon 1007 or the equivalent), or 10 cmdishes such as Fisher 8-757-12 were coated with anti-myc mousemonoclonal antibody, diluted to 10 micrograms per ml in 50 mM Tris HClpH 9.5. 3 ml of antibody was used to coat each 6 cm dish or 10 ml wasused per 10 cm dish; plates were exposed to antibody for about 1.5 hrs,then antibody was removed to the next dish, allowed to stand for 1.5hrs, and then removed again to a third dish. Plates were washed threetimes with 0.15M NaCl (a wash bottle is convenient for this), andincubated with 3 ml 1 mg/ml BSA in PBS overnight. In particular"panning" was performed as follows: cells were cultured in 100 mmdishes. Medium was aspirated from each dish, and 2 ml PBS/0.5 mMEDTA/0.02% azide was added and the mixture was incubated at 37° for 30min to detach cells from the dish. The cells were triturated vigorouslywith a short pasteur pipet, collected from each dish in a centrifugetube, and spun 4 min at a setting of 2.5 (200×g). Cells were resuspendedin 0.5-1.0 ml PBS/EDTA/azide/5% FBS and incubated with CNTF/myc for 30min on ice. An equal volume of PBS/EDTA/azide was added, layeredcarefully on 3 ml PBS/EDTA/azide/2% Ficoll, spun 4 min at a setting of2.5, and the supernatant was aspirated in one smooth movement. The cellswere then incubated with 9E10 antibody for 30 minutes on ice, and thespin through PBS/EDTA/azide/2% Ficoll was repeated. The cells were takenup in 0.5 ml PBS/EDTA/azide and aliquots were added to anti-myc mousemonoclonal antibody-coated dishes containing 3 ml PBS/EDTA/azide/5% FBS.Cells were added from at most two 60 mm dishes to one 60 mm antibodycoated plate, and allowed to sit at room temperature 1-3 hours. Excesscells not adhering to dish were removed by gentle washing with PBS/5%serum or with medium (2 or 3 washes of 3 ml were usually sufficient).

6.1.3. Identification of Clones Containing The Ciliary NeurotrophicFactor Receptor Gene

Plasmid DNA from the expression library was transfected into COSM5 cells(approximately 250-500 ng per 100 mm dish; 2 dishes were transfected),using DEAE/chloroquine according to standard procedures. Two days aftertransfection, cells were detached from their dishes and subjected to theAruffo/Seed panning procedure modified as described supra.

After washing nonadhering cells from the plates, Hirt supernatants(Hirt, 1967, J. Mol. Biol. 26:365-369) were prepared, and plasmid DNAwas precipitated in the presence of 10-20 μg of tRNA. The resulting DNAwas introduced into DH10B bacteria (Electromax, BRL) by electroporationaccording to the manufacturer's instructions. Cultures grown from theelectroporated bacteria were used to prepare plasmid DNA for anotherround of transfection and panning; a plate of COS cells transfected withthis plasmid DNA clearly revealed a large number of COS cells expressingthe CNTFR by an indirect iodinated-antibody binding assay (see FIGS. 1B1and 1B2 for representative data, see below for assay methods). After asecond round of panning/plasmid DNA isolation/electroporation on thesetransfectants, the bacterial transformants resulting from theelectroporation step were plated out on ampicillin plates. Individualbacterial colonies were picked, and plasmid DNA prepared from each ofthe clones was transfected individually into COS cells for assay. Out of15 plasmids tested, 14 resulted in transfected COS cells expressing CNTFbinding sites by a variety of assays, including the indirect antibodybinding assay and fluorescence activated cell sorting (FACS) analysisdescribed infra.

6.1.4. Direct ¹²⁵ I-hCNTF Binding Assay

COS cells were transfected with plasmid DNA from the library, theenriched library, or individual clones. After 48 hours, the media wasremoved and replaced with 0.25 ml of binding buffer (RPMl1640 with 10%FBS and 0.1% NaN₃) containing ¹²⁵ I-hCNTF alone or with unlabelledhCNTF. Incubations with ¹²⁵ I-hCNTF were for 60 minutes at roomtemperature. After incubations were complete, the ¹²⁵ I-hCNTF solutionwas removed and the cells were washed three times with 1.0 ml of bindingbuffer and then lysed with 0.25 ml of 0.1N NaOH. This lysate wastransferred to a 12×75 mm polystyrene tube and placed in a gammacounter. Non-specific binding was determined by the addition of at least100 fold excess unlabelled hCNTF. After the last wash the plates wereautoradiographed.

6.1.5. Fluorescence Activated Cell-Sorting Analysis

Transfected COS cells were incubated sequentially with CNTF/myc, 9E10antibody, and FITC-labelled goat anti-mouse antibody. Then they weredetached from dishes and subjected to FACS analysis. The results oftransfections with a negative and positive plasmid are depicted in FIGS.1D (i) and 1D (ii) COS cells transfected with a CNTF-receptor expressingplasmid contain a large subpopulation displaying greatly increasedfluorescence by this assay.

6.1.6. Iodination of hCNTF

10 μg hCNTF (560 pg/ml in 10 mM NaPO₄ pH7.4) was iodinated with 1 mCi¹²⁵ INa using lactoperoxidase 6 ng/μl (Sigma) for 15 minutes at 20° C.After 15 minutes the reaction was quenched with an equal volume ofbuffer containing 0.1M Nal, 0.1% BSA and 0.1% cytochrome C, 0.3% HOAc,0.05% phenol red and 0.02% NaN3. Aliquots were removed for determinationof TCA precipitatable counts. The remainder was loaded onto a BioRadPD-10 biogel column equilibrated with 0.05M NaPO₄, 0.1M NaCl, 0.5 mg/mlprotamine sulfate and 1 mg/ml BSA. Fractions were collected and TCAprecipitatable counts determined.

6.1.7. Sequencing of CNTFR

Sequencing was performed using a kit (U.S. Biochemical) for dideoxydouble stranded DNA using Sequenase™, according to the manufacturer'sinstructions.

6.1.8. Indirect ¹²⁵ I Goat Anti-Mouse Antibody Binding Assay

COS cells were transfected with plasmid DNA from the library, theenriched library, or individual clones. After 48 hours, cells wereincubated sequentially for 30 minutes on ice with PBS (with Ca, Mg)/5%FBS containing:

1) 1 μg/ml CNTF-myc

2) 10 μg/ml 9E10;

3) ¹²⁵ I goat anti-mouse antibody (GaM) (0.5-1 μCi/ml).

Cells were washed 3×5 minutes in PBS/5% FBS after each step. After thelast wash, the plates were autoradiographed.

For the individual clones, a quantitative estimate of totalradioactivity bound was made with a hand-held gamma counter.

6.2. Results and Discussion 6.2.1. Restriction Analysis

On restriction analysis, the 14 positive clones fell into four classes:

a) I2=I7 (2kb)

b) I1=I5=I6 (2kb)

c) I4=I8=I9=I11=I14=I15 (4kb)

d) I10=I12=I13 (1.6kb) (I3 was negative))

Members of each class produced an identical pattern of bands ondigestion with the enzyme PstI. Further restriction analysis revealedthat the four classes of clones overlapped, and preliminary sequencedata confirmed that they shared overlapping sequences at their 5' ends.Curiously, class (b) proved to have its insert in the wrong orientationin the vector with respect to the eukaryotic promoter element. As can beseen from Table I, these clones were low expressors relative to theother clones. Transcription in these clones may arise from a weakcryptic promoter in the region downstream of the vector's polylinker.

6.2.2. In Vitro Transcription and Translation

To characterize the proteins coded for by the four classes of clones,they were all transcribed from the T7 promoter in the 5' region of thevector polylinker. After in vitro translation, the products wereelectrophoresed on a polyacrylamide gel. Class (a) produced no protein,since it is in the wrong orientation with respect to the T7 promoter.The other three classes all produced proteins of identical sizes(approximately 42 kd), verifying that they encoded the same protein.

                  TABLE I    ______________________________________    Quantitation of .sup.125 I-GaM binding in CNTFR clones.    Clone           CPM bound    ______________________________________    I1              2000    I2              8500    I3               600    I4              9000    I5              2000    I6              1600    I7              6000    I8              7500    I9              7000     I10            4500     I11            7000     I12            5000     I13            8000     I14            10000     I15            8000    Negative Control                     500    Background       250    ______________________________________

6.2.3. Binding Analysis with CNTF

The results of the indirect CNTF-myc binding assay using 9E10 anti-mycantibody and ¹²⁵ I goat anti-mouse antibody are shown in FIGS. 1B1, 1B2,1C1, and 1C2 as well as in Table I. In (FIGS. 1B2) the plate on the leftresults from transfection of the unenriched library, while the plate onthe right is (FIG.1B2) from transfection of the enriched library plasmidDNA rescued after one round of panning (using approximately the sameamount of DNA as for the unenriched library). Note the large number ofdark spots seen only in the plate on the right (FIG. 1B2), eachrepresenting a single COS cell expressing CNTF-myc binding site detectedby radioautography.

For the individual positive clones discussed in Section 6.2.1, aquantitative estimate of total radioactivity was made with a hand-heldgamma counter. The results of this assay for the individual clonesI1-I15 are shown in Table I and demonstrate that 14 out of 15 clonesexpress CNTF binding sites, as determined by indirect antibody bindingassay. In addition, fragments of the plates from some of the individualclones were autoradiographed, as shown in FIGS. 1C1 and 1C2.

A second demonstration of indirect binding utilized CNTF-myc followed by9E10 body, FITC-labelled goat anti-mouse antibody, and FACS analysis, asshown in FIGS. 1D1 and 1D2. COS cells transfected with positive clonesdemonstrated a 100-fold increase in expression of CNTFR as compared withcells transfected with negative clones.

The indirect binding data obtained using CNTF-myc was verified usingdirect ¹²⁵ I-CNTF binding, as shown in Table II. The receptor expressedon transfected COS cells specifically binds to iodinated CNTF as well asto the CNTF-myc ligand, as did the SH-SY5Y cells from which the CNTFRwas cloned. Each transfected COS cell expresses about 30-fold morereceptor per cell than SH-SY5Y cells.

                  TABLE II    ______________________________________    Binding Analysis With Iodinated CNTF    COS I2                  SH-SY5Y    Conc.  Specific             Specific    .sup.125 I-CNTF           cpm bound cpm/cell*  cpm bound                                        cpm/cell    ______________________________________    2.16 nM           1412      2.17 × 10.sup.-2                                1284    4.28 × 10.sup.-3    ______________________________________     Monolayer binding assays were performed in 24 well culture plates using 3     × 10.sup.5 SHSY5Y cells/well or 6.5 × 10.sup.4 COS cells/well     Specific cpm bound was calculated by subtracting cpm bound in the presenc     of 1000fold excess of unlabelled CNTF from the cpm bound only in the     presence of .sup.125 ICNTF at the concentration indicated. No specific     binding was detected in untransfected COS cells.     *COS cells were assayed 48 hours after transfection by DEAE Dextran in     which typically only 20-40% of the cells are transfected. Assuming 20% CO     cells are transfected, the specific cpm bound indicate that each     transfected COS cell expresses about 30fold more receptors per cell than     SHSY5Y cells.

6.2.4. Sequence of CNTFR and Homology to Other Growth Factor Receptors

The CNTFR contains motifs which are shared with a variety of otherreceptors. The extracellular portion of the CNTFR contains both an"immunoglobulin" domain at its N-terminus, as well as a "cytokinereceptor" domain which is separated from the "immunoglobulin" domain bya short hinge region. Although many receptors have homology to eitherthe "immunoglobulin" (SEQ ID NOS. 2,8,9,10,11,12,13) or "cytokinereceptor" domains (FIGS. 3A and 3B), only one receptor- the IL-6receptor- shares the same particular arrangement of these domains withthe CNTFR (FIGS. 3A and 3B). The IL-6 receptor is thus the protein mostrelated to the CNTFR (FIG. 4). Interestingly, the IL-6 receptor is alsosimilar to the CNTFR in that it has a very short intracytoplasmic domainwhich is apparently not required for initiating responses upon IL-6binding (Hibi et al., 1990, Cell 63:1149-1157). Recently, a novel signaltransducer for the IL-6 receptor, termed gp 130, was molecularly cloned.This transducer does not bind IL-6 by itself, but it does confer highaffinity binding to the IL-6 receptor and it is required to transducethe IL-6 signal (Hibi et al., 1990, Cell 63:1149-1157). Our cloning ofthe CNTFR reveals that it shares important features with the IL-6receptor that are not found in other known receptors, thus defining anew family of receptors. The similarities between IL-6R and CNTFRsuggest that CNTFR is likely to utilize the same signal transducer asthe IL-6 receptor, or a related molecule. Finally, the identification ofCNTFR-related receptors should aid in the identification of novelligands that would bind to these receptors.

7. EXAMPLE: TISSUE LOCALIZATION OF MESSAGE FOR CNTFR 7.1. Materials andMethods 7.1.1. CNTFR Probe Preparation

Molecular cloning of the coding region for hCNTFR into the pCMXexpression vector is described in U.S. patent application entitled"Mammalian Expression Vector" filed concurrently herewith, and theresulting expression vector is depicted in FIG. 6. A PCR probe extendingfrom base 889 to base 1230 of the CNTFR sequences was synthesized andused as a probe for Northern analysis.

7.1.2. RNA Preparation and Northern Blots

Selected tissues were dissected from Sprague-Dawley rats and immediatelyfrozen in liquid nitrogen. RNAs were isolated by homogenization oftissues in 3M LiCl, 6M urea, as described in Bothwell et al. 1990(Methods of Cloning and Analysis of Eukaryotic Genes, Boston, Mass.,Jones and Bartlett). RNAs (10 μg) were fractionated by electrophoresisthrough quadruplicate 1% agarose-formaldehyde gels (Bothwell et al.,1990, Methods of Cloning and Analysis of Eukaryotic Genes, Boston,Mass., Jones and Bartlett) followed by capillary transfer to nylonmembranes (MagnaGraph, Micron Separations Inc.) with 10×SSC (pH 7). RNAswere UV-cross-linked to the membranes by exposure to ultraviolet light(Stratalinker, Stratagen, Inc.) and hybridized at 68° C. withradiolabeled probes in the presence of 0.5M NaPO₄ (pH 7), 1% bovineserum albumin (fraction V, Sigma, Inc.) 7% SDS, 1 mM EDTA (Mahmoudi etal., 1989, Biotechniques 7:331-333), 100 μg/ml sonicated, denaturedsalmon sperm DNA. Filters were washed at 68° C. with 3×SSC, 0.1% SDS andsubjected to autoradiography for 1 day to 2 weeks with one or twointensifying screens (Cronex, DuPont) and X-ray film (SAR-5, Kodak) at70° C. Ethidium bromide staining of the gels demonstrated thatequivalent levels of total RNA were being assayed for the differentsamples (as in Maisonpierre et al., 1990, Science 247:1446-1451.

7.2. Results

As shown in FIG. 5, CNTFR mRNA was detectable in tissues of the centralnervous system at low levels in sciatic nerve and adrenals, and inmuscle. This would indicate that CNTF possesses not only neurotrophicactivity, but myotrophic activity as well, and may explain theinvolvement of both the central nervous system and muscle in certaindisorders, such as Duchennes muscular dystrophy and congenital myotonicdystrophy, in which patients may suffer from mental retardation.Expression of CNTFR in muscle suggests CNTF may have a role in musclephysiology. Thus, in addition to action on neurons, CNTF may haveimportant action in muscle such as functioning as a myotrophic agent, orotherwise effect muscle development and/or differentiation.

8. EXAMPLE: EVIDENCE THAT THE CNTF RECEPTOR IS LINKED TO THE CELLSURFACE VIA A GLYCOSYL-PHOSPHATIDYLINOSITOL (GPI) LINKAGE 8.1. Materialsand Methods

SH-SY5Y cells were cultured in a 24-well plate (Falcon) in RPMIsupplemented with 10% inactivated fetal bovine serum. For experiments inwhich phospholipase (and control) treatments were done prior toCNTF-binding, the media was aspirated, cells were rinsed twice inPBS(+Ca/Mg), and then incubated with PBS(+Ca/Mg) supplemented with orwithout phosphatidylinositol-specific phospholipase (PI-PLC) at finalconcentration of 500 mU/ml (purchased from Boehringer Mannheim,catalogue # 1143-069) for 45 minutes at 37° C. Cells were then washedthree times with binding buffer (PBS(+Ca/Mg) and 5% fetal bovine serum)and then incubated with 250 microliters binding buffer containingiodinated CNTF (approximately 100 picomolar) with or without athousand-fold excess of unlabelled CNTF for 30 minutes at roomtemperature. For experiments in which the iodinated CNTF was bound priorto PI-PLC treatment, cells were first incubated in binding buffercontaining iodinated CNTF with or without excess unlabelled CNTF at 37°C. for 45 minutes. Cells were then washed two times with PBS(+Ca/Mg) andthen incubated for 45 minutes with PBS(+Ca/Mg) supplemented with orwithout PI-PLC (final concentration 500 mU/ml). Cells were then rinsedthree times with binding buffer. In all cases cells were solubilizedprior to counting in 0.1N NaOH, and then counted.

8.2. Results and Discussion

The sequence of the CNTF receptor revealed that the encoded proteinended within a hydrophobic region that followed the extra-cytoplasmicdomains, without any apparent stop transfer sequence orintra-cytoplasmic domain. This structure seemed reminiscent of theC-terminals found on membrane proteins which lack transmembrane domainsand are attached to the cell surface via GPI-linkages (Ferguson andWilliams, 1988). Thus, experiments were performed to test whether theCNTF receptor was linked to the cell surface via a GPI-linkage. As shownin Table III, treatment of SH-SY5Y cells with PI-PLC completelyeliminated the ability of SH-SY5Y cells to subsequently bind CNTF,consistent with the notion that the CNTF receptor is linked to the cellsurface via a GPI-linkage. However, CNTF already bound to SH-SY5Y cellscannot be released by PI-PLC treatment (Table III). Interestingly, asoluble form of the IL-6 receptor can tightly associate with a secondmembrane protein (GP130) required for IL-6 signal transduction. Thus,prevention of CNTF receptor release by prior binding to CNTF may be dueto an association between the CNTF, its receptor, and its signaltransducer (GP130 or a GP130 analog). Alternatively, CNTF-binding mayalter the structure of the CNTF receptor, making it less susceptible toPI-PLC (several GPI-linked proteins have PI-PLC resistant forms).

The finding that the CNTF receptor is attached to the cell surface via aGPI-linkage has important ramifications. It represents the first knowngrowth factor receptor to be linked to the membrane in this fashion,raising the possibility that additional receptors may be GPI-linked.Because several proteins have both GPI-linked forms as well as formsthat contain conventional transmembrane domains, our findings raise thepossibility that the CNTF receptor has an alternative C-terminus thatcould encode a transmembrane domain, and similarly that the IL-6receptor has a GPI-linked form. The GPI-linked forms of growth factorreceptors may be able to utilize novel mechanisms of receptor regulationand release. For example, down-regulation of surface receptors couldrapidly occur by releasing the GPI-linked receptors by activatingextra-cytoplasmic phospholipase activities. These released receptorsmight also act on other cells, either alone or when bound to CNTF inmuch the same way that soluble IL-6 receptor has been shown to bind IL-6and activate cells expressing GP130.

The possibility that release of CNTF receptors using PI-PLC could blockCNTF action may have important implications. It could be used to verifythat observed effects of CNTF are due to the cloned CNTF receptor.Therapeutically, PI-PLC could be used to release CNTF receptors andpossibly block CNTF action in cases where CNTF activity is thought to bedetrimental.

If the CNTF-blockable PI-PLC release of the CNTF receptor is due to theformation of a tertiary complex between the CNTF, its receptor, and thepotential signal transducing protein, then this feature of the receptorcould be used to define and molecularly clone the transducing molecule.

                  TABLE III    ______________________________________    Analysis Of PI-PLC Treatment On CNTF Binding To SH-SY5Y Cells                   CPM Bound                   No Cold Excess                             Cold Excess    ______________________________________    Pre-Treat with PI-PLC    No PI-PLC        1440        370    With PI-PLC       420        310    Bind CNTF Before PI-PLC    No PI-PLC        1250        310    With PI-PLC      1060        300    ______________________________________

9. The Effects of CNTF on Denervated Rat Skeletal Muscle in Vivo

The goal of the experiments described herein was to examine the effectsof purified recombinant CNTF on denervated skeletal muscle in vivo andto determine whether CNTF could prevent some of the phenotypic changesassociated with denervation atrophy such as muscle weight andmyofibrillar protein loss. We found that the CNTF receptor is expressedin skeletal muscle on both myotubes and myoblasts, and that CNTFprevents the loss of both muscle weight and myofibril protein contentassociated with denervation atrophy.

9.1. The CNTF Receptor is Expressed in Skeletal Muscle on Both Myotubesand Myoblasts

Northern blot analysis was performed on RNA samples derived from avariety of rat tissues in order to identify the primary cellular targetsof CNTF as shown in FIG. 5. A probe derived from the human CNTF receptorcoding region identified a 2 kb transcript whose expression wasgenerally restricted to the central nervous system, except forsurprisingly high levels found in skeletal muscle and low levels inadrenal gland and sciatic nerve.

A more detailed analysis of the CNTF receptor expression specifically inskeletal muscle was carried out as described supra by Northern blottingusing MRNA prepared from specific rat muscle types, purified humanmuscle myotubes, and several skeletal muscle cell lines of both mouseand rat origin (FIG. 7). Using a human probe for the CNTF receptor, twomRNA species (2.0 and 1.7 kb) were detected in several muscle RNAsamples. FIG. 7 demonstrates that the CNTF receptor is expressed in bothmyotube and myoblast muscle cell lines of either mouse (lanes 1 and 2)or rat (lanes 3 and 4) origin, as well as in both red slow-twitch soleusmuscle and white fast-twitch extensor digitorum longus (EDL) muscle ofthe rat (lanes 5 and 6, respectively). It appeared that the level ofCNTF receptor mRNA was increased in both soleus (lane 12) and EDL muscle(lane 14) that were first denervated for 72 hours relative to theirsham-operated contralateral controls (lanes 11 and 13 respectively).Interestingly, the highest level of expression was observed in RNAsamples from myotubes derived from human fetal skeletal muscle. Thesemyotubes were cultured and then purified away from fibroblasts and othernon-muscle cells by fluorescence-activated cell sorting prior to RNAisolation (lane 8). We noted that two distinct CNTF receptor mRNAspecies were identified on the muscle cell Northern blot and that the1.7 kb CNTF receptor message was preferentially expressed in themyoblast cell line C2C12 mb (lane 1) and may represent an alternativelyspliced form of the receptor.

9.2. CNTF Prevents the Loss of Both Muscle Weight and MyofibrillarProtein Content Associated with Denervation Atrophy 9.2.1. DenervationSurgery

The various animal groups used for these studies are described in TableIV, infra. Generally, three animals comprised a single group. For allexperimental groups, an initial incision of approximately 20 cm was madethrough the skin of the right hindlimb at midthigh level. Following thissurgical procedure, the 20 cm cut was also performed on the lefthindlimb at midthigh in order to carry out the sham-operation.

In animal groups 2-6, the soleus muscle of the right hindlimb wasdenervated by surgically removing a 2-5 mm segment of the right sciaticnerve at midthigh level to leave a distal nerve stump of 32 to 35 mm(labeled as A in FIG. 8). The left soleus muscle served as the controlin that a sham-operation was performed on this muscle by gently pullingon the sciatic nerve 32 to 35 mm from its point of innervation of thesoleus muscle. All surgeries were carried out while the animals wereunder light chloro-pentobarbitol anesthesia (0.3 g/kg). Animal group 1(controls) did not receive any denervation and were not injected. Allanimals weighed between 100 and 150 grams.

9.2.2. Treatments

Animals in groups 1 and 2 were not treated. Animals in group 3 wereinjected daily for a total of 4 days intramuscular (IM) withphosphate-buffered saline (PBS) containing 1 mg/ml of BSA (PBS/BSA).Mulwiple injections were made into the muscles of the midthigh on bothsides of the animals. Animals in group 4 were injected daily for 4 daysIM with recombinant rat CNTF (1 mg/kg) containing 1 mg/ml of BSA(CNTF/BSA). Multiple injections were made on both sides of the animal asdescribed above. Animals in group 5 were also injected daily withrCNTF/BSA but subcutaneously (SC) rather than IM. Animals in group 6were injected daily (SC) with PBS/BSA.

                  TABLE IV    ______________________________________                    Surgical   Denervation    Group  # Animals                    Protocol   Time     Treatment    ______________________________________    1      3        None       96 hours None    2      3        R-Den/L-Sham                               96 hours None    3      3        R-Den/L-Sham                               96 hours PBS/BSA                                        (1 mg/ml)    4      3        R-Den/L-Sham                               96 hours CNTF/BSA                                        (1 mg/kg) (IM)    5      3        R-Den/L-Sham                               96 hours CNTF/BSA                                        (1 mg/kg) (SC)    6      3        R-Den/L-Sham                               96 hours PBS/BSA (SC)    ______________________________________     R-Den = right hindlimb denervated;     LSham = left hindlimb shamoperated

9.2.3. Muscle Weight and Protein Analysis

96 hours after the denervation surgery was performed, the animals weresacrificed by decapitation, and the soleus muscles were carefullyexcised from tendon to tendon. The soleus muscles were placed on a weighboat on ice, tendons were removed with a scalpel, and the muscles werethen weighed immediately so as to prevent any drying. To preparemyofibrillar protein homogenates, the excised soleus muscles werepooled, minced while on ice in a cold room, and then homogenized in PBScontaining 0.32M sucrose and 3 mM MgCl₂ (2.5% w/v). The homogenate wascentrifuged at approximately 800×g and the supernatants were assayed fortotal myofibril protein per muscle by using the Bio-Rad Dye Bindingprocedure according to the manufacturers recommendations.

FIG. 9 demonstrates that denervated soleus muscle decreasedsignificantly (p<0.01) in wet weight approximately 25% at 96 hours.Daily injection of PBS/BSA had no effect on this denervation-dependentmuscle weight loss. However, denervated soleus muscles from ratsinjected daily with CNTF (1 mg/kg)/BSA weighed approximately 5% lessthan their contralateral sham-operated controls. The wet weights of theCNTF treated denervated and sham-operated soleus muscles were notsignificantly different from unoperated controls. CNTF, when injected SCdaily for 4 days (group 5), also appeared to significantly prevent thedenervation-induced loss of myofibrillar protein, and the loss ofprotein paralleled the decrease in muscle wet weight (Table V).

                  TABLE V    ______________________________________    Effect Of CNTF On Denervated Soleus Muscle Protein Content                   Total Myofibril Protein    Muscle Sample  (mg per Muscle)                                  % of Sham    ______________________________________    Group 1 - No denervation                   7.5    Group 2 - den. - no injection                   5.8            80    sham - no injection                   7.2    Group 3 - dem + PBS (IM)                   5.2            75    sham + PBS (IM)                   6.9    Group 4 - den + CNTF (IM)                   6.5            83    sham + CNTF (IM)                   7.8    Group 5 - den + CNTF (SC)                   6.6            93    sham + CNTF (SC)                   7.1    Group 6 - den + PBS (SC)                   5.3            67    sham + PBS (SC)                   7.9    ______________________________________     (IM) = intramuscular injection;     (SC) = subcutaneous injection;     data presented represent the total protein content of 3 pooled soleus     muscles

When injected daily IM, a less pronounced effect of CNTF on totalmyofibril protein was observed.

We found that the CNTF receptor is expressed in skeletal muscle on bothmyotubes and myoblasts, and that CNTF prevents the loss of both muscleweight and myofibril protein content associated with denervationatrophy.

10. DEPOSIT OF MICROORGANISM

The following deposit has been made on Mar. 26, 1991 with TheAgricultural Research Culture Collection (NRRL), 1815 North UniversityStreet, Peoria, Ill., 61604:

E. coli carrying plasmid pCMX-hCNTFR (I2), an expression plasmidcomprising hCNTFR encoding sequences, assigned accession number NRRLB-18789.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

The present invention is not to be limited in scope by the constructdeposited or the embodiments disclosed in the examples which areintended as illustrations of a few aspects of the invention and anyembodiments which are functionally equivalent are within the scope ofthis invention. Indeed, various modifications of the invention inaddition to those shown and described herein will become apparent tothose skilled in the art and are intended to fall within the scope ofthe appended claims.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 15    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1591 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 289..1404    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    CCTCGAGATCCATTGTGCTCAAAGGGCGGCGGCAGCGGAGGCGGCGGCTCCAGCCGGCGC60    GGCGCGAGGCTCGGCGGTGGGATCCGGCGGGCGGTGCTAGCTCCGCGCTCCCTGCCTCGC120    TCGCTGCCGGGGGCGGTCGGAAGGCGCGGCGCGAAGCCCGGGTGGCCCGAGGGCGCGACT180    CTAGCCTTGTCACCTCATCTTGCCCCCTTGGTTTTGGAAGTCCTGAAGAGTTGGTCTGGA240    GGAGGAGGAGGACATTGATGTGCTTGGTGTGTGGCCAGTGGTGAAGAGATGGCTGCT297    MetAlaAla    CCTGTCCCGTGGGCCTGCTGTGCTGTGCTTGCCGCCGCCGCCGCAGTT345    ProValProTrpAlaCysCysAlaValLeuAlaAlaAlaAlaAlaVal    51015    GTCTACGCCCAGAGACACAGTCCACAGGAGGCACCCCATGTGCAGTAC393    ValTyrAlaGlnArgHisSerProGlnGluAlaProHisValGlnTyr    20253035    GAGCGCCTGGGCTCTGACGTGACACTGCCATGTGGGACAGCAAACTGG441    GluArgLeuGlySerAspValThrLeuProCysGlyThrAlaAsnTrp    404550    GATGCTGCGGTGACGTGGCGGGTAAATGGGACAGACCTGGCCCCTGAC489    AspAlaAlaValThrTrpArgValAsnGlyThrAspLeuAlaProAsp    556065    CTGCTCAACGGCTCTCAGCTGGTGCTCCATGGCCTGGAACTGGGCCAC537    LeuLeuAsnGlySerGlnLeuValLeuHisGlyLeuGluLeuGlyHis    707580    AGTGGCCTCTACGCCTGCTTCCACCGTGACTCCTGGCACCTGCGCCAC585    SerGlyLeuTyrAlaCysPheHisArgAspSerTrpHisLeuArgHis    859095    CAAGTCCTGCTGCATGTGGGCTTGCCGCCGCGGGAGCCTGTGCTCAGC633    GlnValLeuLeuHisValGlyLeuProProArgGluProValLeuSer    100105110115    TGCCGCTCCAACACTTACCCCAAGGGCTTCTACTGCAGCTGGCATCTG681    CysArgSerAsnThrTyrProLysGlyPheTyrCysSerTrpHisLeu    120125130    CCCACCCCCACCTACATTCCCAACACCTTCAATGTGACTGTGCTGCAT729    ProThrProThrTyrIleProAsnThrPheAsnValThrValLeuHis    135140145    GGCTCCAAAATTATGGTCTGTGAGAAGGACCCAGCCCTCAAGAACCGC777    GlySerLysIleMetValCysGluLysAspProAlaLeuLysAsnArg    150155160    TGCCACATTCGCTACATGCACCTGTTCTCCACCATCAAGTACAAGGTC825    CysHisIleArgTyrMetHisLeuPheSerThrIleLysTyrLysVal    165170175    TCCATAAGTGTCAGCAATGCCCTGGGCCACAATGCCACAGCTATCACC873    SerIleSerValSerAsnAlaLeuGlyHisAsnAlaThrAlaIleThr    180185190195    TTTGACGAGTTCACCATTGTGAAGCCTGATCCTCCAGAAAATGTGGTA921    PheAspGluPheThrIleValLysProAspProProGluAsnValVal    200205210    GCCCGGCCAGTGCCCAGCAACCCTCGCCGGCTGGAGGTGACGTGGCAG969    AlaArgProValProSerAsnProArgArgLeuGluValThrTrpGln    215220225    ACCCCCTCGACCTGGCCTGACCCTGAGTCTTTTCCTCTCAAGTTCTTT1017    ThrProSerThrTrpProAspProGluSerPheProLeuLysPhePhe    230235240    CTGCGCTACCGACCCCTCATCCTGGACCAGTGGCAGCATGTGGAGCTG1065    LeuArgTyrArgProLeuIleLeuAspGlnTrpGlnHisValGluLeu    245250255    TCCGACGGCACAGCACACACCATCACAGATGCCTACGCCGGGAAGGAG1113    SerAspGlyThrAlaHisThrIleThrAspAlaTyrAlaGlyLysGlu    260265270275    TACATTATCCAGGTGGCAGCCAAGGACAATGAGATTGGGACATGGAGT1161    TyrIleIleGlnValAlaAlaLysAspAsnGluIleGlyThrTrpSer    280285290    GACTGGAGCGTAGCCGCCCACGCTACGCCCTGGACTGAGGAACCGCGA1209    AspTrpSerValAlaAlaHisAlaThrProTrpThrGluGluProArg    295300305    CACCTCACCACGGAGGCCCAGGCTGCGGAGACCACGACCAGCACCACC1257    HisLeuThrThrGluAlaGlnAlaAlaGluThrThrThrSerThrThr    310315320    AGCTCCCTGGCACCCCCACCTACCACGAAGATCTGTGACCCTGGGGAG1305    SerSerLeuAlaProProProThrThrLysIleCysAspProGlyGlu    325330335    CTGGGCAGCGGCGGGGGACCCTGCGCACCCTTCTTGGTCAGCGTCCCC1353    LeuGlySerGlyGlyGlyProCysAlaProPheLeuValSerValPro    340345350355    ATCACTCTGGCCCTGGCTGCCGCTGCCGCCACTGCCAGCAGTCTCTTG1401    IleThrLeuAlaLeuAlaAlaAlaAlaAlaThrAlaSerSerLeuLeu    360365370    ATCTGAGCCCGGCACCCCATGAGGACATGCAGAGCACCTGCAGAGGAGCAGGA1454    Ile    GGCCGGAGCTGAGCCTGCAGACCCCGGTTTCTATTTTGCACACGGGCAGGAGGACCTTTT1514    GCATTCTCTTCAGACACAATTTGTGGAGACCCCGGCGGGCCCGGGCCTGCCGCCCCCCAG1574    CCCTGCCGCACCAAGCT1591    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 372 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    MetAlaAlaProValProTrpAlaCysCysAlaValLeuAlaAlaAla    151015    AlaAlaValValTyrAlaGlnArgHisSerProGlnGluAlaProHis    202530    ValGlnTyrGluArgLeuGlySerAspValThrLeuProCysGlyThr    354045    AlaAsnTrpAspAlaAlaValThrTrpArgValAsnGlyThrAspLeu    505560    AlaProAspLeuLeuAsnGlySerGlnLeuValLeuHisGlyLeuGlu    65707580    LeuGlyHisSerGlyLeuTyrAlaCysPheHisArgAspSerTrpHis    859095    LeuArgHisGlnValLeuLeuHisValGlyLeuProProArgGluPro    100105110    ValLeuSerCysArgSerAsnThrTyrProLysGlyPheTyrCysSer    115120125    TrpHisLeuProThrProThrTyrIleProAsnThrPheAsnValThr    130135140    ValLeuHisGlySerLysIleMetValCysGluLysAspProAlaLeu    145150155160    LysAsnArgCysHisIleArgTyrMetHisLeuPheSerThrIleLys    165170175    TyrLysValSerIleSerValSerAsnAlaLeuGlyHisAsnAlaThr    180185190    AlaIleThrPheAspGluPheThrIleValLysProAspProProGlu    195200205    AsnValValAlaArgProValProSerAsnProArgArgLeuGluVal    210215220    ThrTrpGlnThrProSerThrTrpProAspProGluSerPheProLeu    225230235240    LysPhePheLeuArgTyrArgProLeuIleLeuAspGlnTrpGlnHis    245250255    ValGluLeuSerAspGlyThrAlaHisThrIleThrAspAlaTyrAla    260265270    GlyLysGluTyrIleIleGlnValAlaAlaLysAspAsnGluIleGly    275280285    ThrTrpSerAspTrpSerValAlaAlaHisAlaThrProTrpThrGlu    290295300    GluProArgHisLeuThrThrGluAlaGlnAlaAlaGluThrThrThr    305310315320    SerThrThrSerSerLeuAlaProProProThrThrLysIleCysAsp    325330335    ProGlyGluLeuGlySerGlyGlyGlyProCysAlaProPheLeuVal    340345350    SerValProIleThrLeuAlaLeuAlaAlaAlaAlaAlaThrAlaSer    355360365    SerLeuLeuIle    370    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 55 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    ValThrLeuThrCysXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaVal    151015    HisTrpValLeuArgLysXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa    202530    XaaXaaXaaXaaXaaArgLeuLeuLeuArgSerValGlnLeuHisAsp    354045    SerGlyAsnTyrSerCysTyr    5055    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 46 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    LeuAsnLeuSerCysXaaXaaXaaXaaXaaXaaXaaXaaXaaTyrSer    151015    TrpArgIleXaaXaaXaaXaaXaaXaaXaaXaaXaaValLeuPheIle    202530    AlaLysIleThrProAsnAsnAsnGlyThrTyrAlaCysPhe    354045    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 62 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    IleThrIleArgCysXaaXaaXaaXaaXaaXaaXaaXaaXaaPheGln    151015    TrpThrTyrProArgMetXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa    202530    XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaIleLeuHisIle    354045    ProThrAlaGluLeuSerAspSerGlyThrTyrThrCysAsn    505560    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 60 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    AlaGlnIleValCysXaaXaaXaaXaaXaaXaaXaaXaaPheAspVal    151015    SerLeuArgHisXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa    202530    XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaThrLeuAsnLeuAspHis    354045    ValSerPheGlnAspAlaGlyAsnTyrSerCysThr    505560    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 54 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    ValThrLeuThrCysXaaXaaXaaXaaXaaXaaXaaXaaPheGlnLeu    151015    ArgArgXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa    202530    XaaXaaXaaXaaPhePheHisLeuAsnAlaValAlaLeuGlyAspGly    354045    GlyHisTyrThrCysArg    50    (2) INFORMATION FOR SEQ ID NO:8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 188 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    CysPheArgLysSerProLeuSerAsnValValCysGluTrpXaaXaa    151015    XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa    202530    XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaCysXaaXaaXaa    354045    XaaXaaXaaXaaXaaXaaXaaCysXaaXaaXaaXaaXaaXaaXaaXaa    505560    XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa    65707580    XaaXaaXaaXaaXaaXaaXaaXaaPheGlnGlyCysGlyIleLeuGln    859095    ProAspProProAlaAsnIleThrValThrAlaValAlaArgAsnPro    100105110    ArgTrpLeuSerValThrTrpXaaXaaXaaXaaXaaXaaXaaXaaXaa    115120125    XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa    130135140    XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa    145150155160    XaaXaaXaaXaaXaaXaaXaaXaaXaaValValGlnLeuArgAlaGln    165170175    GluGluPheGlyGlnGlyGluTrpSerGluTrpSer    180185    (2) INFORMATION FOR SEQ ID NO:9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 185 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    CysArgSerProAspLysGluThrPheThrCysTrpTrpXaaXaaXaa    151015    XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa    202530    XaaXaaXaaXaaXaaXaaXaaCysXaaXaaXaaXaaXaaXaaXaaXaa    354045    XaaXaaCysXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa    505560    XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa    65707580    XaaXaaXaaXaaValAspValThrTyrIleValGluProGluProPro    859095    ArgAsnLeuThrLeuGluValLysGlnLeuLysAspLysLysThrTyr    100105110    LeuTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa    115120125    XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa    130135140    XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa    145150155160    XaaXaaXaaXaaXaaXaaXaaXaaXaaValGlnThrArgCysLysPro    165170175    AspHisGlyTyrTrpSerArgTrpSer    180185    (2) INFORMATION FOR SEQ ID NO:10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 185 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    CysPheThrGlnArgLeuGluAspLeuValCysPheTrpXaaXaaXaa    151015    XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa    202530    XaaXaaXaaXaaXaaXaaCysXaaXaaXaaXaaXaaXaaXaaXaaXaa    354045    XaaXaaXaaXaaXaaXaaCysXaaXaaXaaXaaXaaXaaXaaXaaXaa    505560    XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa    65707580    XaaXaaXaaXaaIleHisIleAsnGluValValLeuLeuAspAlaPro    859095    AlaGlyLeuLeuAlaArgArgAlaGluGluGlySerHisValValLeu    100105110    ArgTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa    115120125    XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa    130135140    XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa    145150155160    XaaXaaXaaXaaXaaXaaXaaValArgAlaArgMetAlaGluProSer    165170175    PheSerGlyPheTrpSerAlaTrpSer    180185    (2) INFORMATION FOR SEQ ID NO:11:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 189 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    CysPheTyrAsnSerArgAlaAsnIleSerCysValTrpXaaXaaXaa    151015    XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa    202530    XaaXaaXaaXaaXaaXaaCysXaaXaaXaaXaaXaaXaaXaaXaaXaa    354045    XaaXaaCysXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa    505560    XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa    65707580    XaaXaaXaaXaaXaaXaaXaaXaaPheLysProPheGluAsnLeuArg    859095    LeuMetAlaProIleSerLeuGlnValValHisValGluThrHisArg    100105110    CysAsnIleSerTrpXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa    115120125    XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa    130135140    XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa    145150155160    XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaValArgValLys    165170175    ProLeuGlnGlyGluPheThrThrTrpSerProTrpSer    180185    (2) INFORMATION FOR SEQ ID NO:12:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 184 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    CysPheSerAspTyrIleArgThrSerThrCysGluTrpXaaXaaXaa    151015    XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa    202530    XaaXaaXaaXaaXaaXaaXaaXaaXaaCysXaaXaaXaaXaaXaaXaa    354045    XaaXaaXaaXaaXaaCysXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa    505560    XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa    65707580    XaaXaaPheSerProSerGlyAsnValLysProLeuAlaProAspAsn    859095    LeuThrLeuHisThrAsnValSerAspGluTrpLeuLeuThrTrpXaa    100105110    XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa    115120125    XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa    130135140    XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa    145150155160    XaaXaaXaaXaaXaaXaaXaaXaaValArgValArgSerGlnIleLeu    165170175    ThrGlyThrTrpSerGluTrpSer    180    (2) INFORMATION FOR SEQ ID NO:13:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 185 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    CysPheIleTyrAsnAlaAspLeuMetAsnCysThrTrpXaaXaaXaa    151015    XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa    202530    XaaXaaXaaXaaXaaXaaXaaCysXaaXaaXaaXaaXaaXaaXaaXaa    354045    XaaXaaXaaXaaCysXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa    505560    XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa    65707580    XaaXaaXaaXaaLeuAspThrLysLysIleGluArgPheAsnProPro    859095    SerAsnValThrValArgCysAsnThrThrHisCysLeuValArgTrp    100105110    XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa    115120125    XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa    130135140    XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa    145150155160    XaaXaaXaaXaaXaaXaaXaaXaaValLysIleArgAlaAlaAspVal    165170175    ArgIleLeuAsnTrpSerSerTrpSer    180185    (2) INFORMATION FOR SEQ ID NO:14:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 34 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:    GACTCGAGTCGACATCGGAGGCTGATGGGATGCC34    (2) INFORMATION FOR SEQ ID NO:15:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 34 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:    CTAAAGACTCCTCCTAGACATCGCCGGCGTATCG34    __________________________________________________________________________

What is claimed is:
 1. A monoclonal antibody that recognizes the ciliaryneurotrophic factor receptor (CNTFR) as depicted in FIG. 2 (SEQ ID NO:2).